Lipid formulated dsrna targeting the pcsk9 gene

ABSTRACT

This invention relates to composition and methods using lipid formulated siRNA targeted to a PCSK9 gene.

CROSS REFERENCE TO RELATED APPLICATIONS

This application a continuation of pending U.S. patent application Ser.No. 13/568,898, filed Aug. 7, 2012 (allowed), which is a continuation ofU.S. patent application Ser. No. 12/816,207, filed Jun. 15, 2010, nowU.S. Pat. No. 8,273,869, issued Sep. 25, 2012, which claims the benefitof U.S. Provisional Application Ser. No. 61/187,169, filed Jun. 15,2009; and U.S. Provisional Application Ser. No. 61/218,350, filed Jun.18, 2009; and U.S. Provisional Application Ser. No. 61/244,790, filedSep. 22, 2009; and U.S. Provisional Application Ser. No. 61/285,598,filed Dec. 11, 2009; and U.S. Provisional Application Ser. No.61/293,474, filed Jan. 8, 2010; and U.S. Provisional Application Ser.No. 61/313,578, filed Mar. 12, 2010, all of which are incorporatedherein by reference, in their entirety, for all purposes.

FIELD OF THE INVENTION

This invention relates to compositions comprising lipid formulated dsRNAtargeting a PCSK9 gene and methods for treating diseases caused by PCSK9gene expression.

REFERENCE TO A SEQUENCE LISTING

This application includes a Sequence Listing submitted electronically asa text file named 24757US_sequencelisting.txt, created on Oct. 18, 2013,with a size of 569,344 bytes. The sequence listing is incorporated byreference.

BACKGROUND OF THE INVENTION

Proprotein convertase subtilisin kexin 9 (PCSK9) is a member of thesubtilisin serine protease family. The other eight mammalian subtilisinproteases, PCSK1-PCSK8 (also called PC1/3, PC2, furin, PC4, PC5/6,PACE4, PC7, and S1P/SKI-1) are proprotein convertases that process awide variety of proteins in the secretory pathway and play roles indiverse biological processes (Bergeron, F. (2000) J. Mol. Endocrinol.24, 1-22, Gensberg, K., (1998) Semin. Cell Dev. Biol. 9, 11-17, Seidah,N. G. (1999) Brain Res. 848, 45-62, Taylor, N. A., (2003) FASEB J. 17,1215-1227, and Zhou, A., (1999) J. Biol. Chem. 274, 20745-20748). PCSK9has been proposed to play a role in cholesterol metabolism. PCSK9 mRNAexpression is down-regulated by dietary cholesterol feeding in mice(Maxwell, K. N., (2003) J. Lipid Res. 44, 2109-2119), up-regulated bystatins in HepG2 cells (Dubuc, G., (2004) Arterioscler. Thromb. Vasc.Biol. 24, 1454-1459), and up-regulated in sterol regulatory elementbinding protein (SREBP) transgenic mice (Horton, J. D., (2003) Proc.Natl. Acad. Sci. USA 100, 12027-12032), similar to the cholesterolbiosynthetic enzymes and the low-density lipoprotein receptor (LDLR).Furthermore, PCSK9 missense mutations have been found to be associatedwith a form of autosomal dominant hypercholesterolemia (Hchola3)(Abifadel, M., et al. (2003) Nat. Genet. 34, 154-156, Timms, K. M.,(2004) Hum. Genet. 114, 349-353, Leren, T. P. (2004) Clin. Genet. 65,419-422). PCSK9 may also play a role in determining LDL cholesterollevels in the general population, because single-nucleotidepolymorphisms (SNPs) have been associated with cholesterol levels in aJapanese population (Shioji, K., (2004) J. Hum. Genet. 49, 109-114).

Autosomal dominant hypercholesterolemias (ADHs) are monogenic diseasesin which patients exhibit elevated total and LDL cholesterol levels,tendon xanthomas, and premature atherosclerosis (Rader, D. J., (2003) J.Clin. Invest. 111, 1795-1803). The pathogenesis of ADHs and a recessiveform, autosomal recessive hypercholesterolemia (ARH) (Cohen, J. C.,(2003) Curr. Opin. Lipidol. 14, 121-127), is due to defects in LDLuptake by the liver. ADH may be caused by LDLR mutations, which preventLDL uptake, or by mutations in the protein on LDL, apolipoprotein B,which binds to the LDLR. ARH is caused by mutations in the ARH proteinthat are necessary for endocytosis of the LDLR-LDL complex via itsinteraction with clathrin. Therefore, if PCSK9 mutations are causativein Hchola3 families, it seems likely that PCSK9 plays a role inreceptor-mediated LDL uptake.

Overexpression studies point to a role for PCSK9 in controlling LDLRlevels and, hence, LDL uptake by the liver (Maxwell, K. N. (2004) Proc.Natl. Acad. Sci. USA 101, 7100-7105, Benjannet, S., et al. (2004) J.Biol. Chem. 279, 48865-48875, Park, S. W., (2004) J. Biol. Chem. 279,50630-50638). Adenoviral-mediated overexpression of mouse or human PCSK9for 3 or 4 days in mice results in elevated total and LDL cholesterollevels; this effect is not seen in LDLR knockout animals (Maxwell, K. N.(2004) Proc. Natl. Acad. Sci. USA 101, 7100-7105, Benjannet, S., et al.(2004) J. Biol. Chem. 279, 48865-48875, Park, S. W., (2004) J. Biol.Chem. 279, 50630-50638). In addition, PCSK9 overexpression results in asevere reduction in hepatic LDLR protein, without affecting LDLR mRNAlevels, SREBP protein levels, or SREBP protein nuclear to cytoplasmicratio.

Loss of function mutations in PCSK9 have been designed in mouse models(Rashid et al., (2005) PNAS, 102, 5374-5379), and identified in humanindividuals (Cohen et al. (2005) Nature Genetics 37:161-165). In bothcases loss of PCSK9 function lead to lowering of total and LDLccholesterol. In a retrospective outcome study over 15 years, loss of onecopy of PCSK9 was shown to shift LDLc levels lower and to lead to anincreased risk-benefit protection from developing cardiovascular heartdisease (Cohen et al., (2006) N. Engl. J. Med., 354:1264-1272).

Recently, double-stranded RNA molecules (dsRNA) have been shown to blockgene expression in a highly conserved regulatory mechanism known as RNAinterference (RNAi). WO 99/32619 (Fire et al.) discloses the use of adsRNA of at least 25 nucleotides in length to inhibit the expression ofgenes in C. elegans. dsRNA has also been shown to degrade target RNA inother organisms, including plants (see, e.g., WO 99/53050, Waterhouse etal.; and WO 99/61631, Heifetz et al.), Drosophila (see, e.g., Yang, D.,et al., Curr. Biol. (2000) 10:1191-1200), and mammals (see WO 00/44895,Limmer; and DE 101 00 586.5, Kreutzer et al.). This natural mechanismhas now become the focus for the development of a new class ofpharmaceutical agents for treating disorders that are caused by theaberrant or unwanted regulation of a gene.

A description of siRNA targeting PCSK9 can be found in U.S. Pat. No.7,605,251 and WO 2007/134161. Additional disclosure can be found in U.S.Patent Publication No. 2010/0010066 and WO 2009/134487

SUMMARY OF THE INVENTION

As described in more detail below, disclosed herein are compositionscomprising lipid formulated siRNA targeting PCSK9, e.g., MC3 formulatedsiRNA targeting PCSK9. Also disclosed are methods of using thecompositions for inhibition of PCSK9 expression and for treatment ofpathologies related to PCSK9 expression, e.g., hyperlipidemia

Accordingly, one aspect of the invention is a compositing comprising anucleic acid lipid particle comprising a double-stranded ribonucleicacid (dsRNA) for inhibiting the expression of a human PCSK9 gene in acell, wherein the nucleic acid lipid particle comprises a lipidformulation comprising 45-65 mol % of a cationic lipid, 5 mol % to about10 mol %, of a non-cationic lipid, 25-40 mol % of a sterol, and 0.5-5mol % of a PEG or PEG-modified lipid, the dsRNA consists of a sensestrand and an antisense strand, and the sense strand comprises a firstsequence and the antisense strand comprises a second sequencecomplementary to at least 15 contiguous nucleotides of SEQ ID NO:1523(5′-UUCUAGACCUGUUUUGCUU-3′), wherein the first sequence is complementaryto the second sequence and wherein the dsRNA is between 15 and 30 basepairs in length.

As described herein the composition includes a cationic lipid. In oneembodiment, the cationic lipid comprises MC3(((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl4-(dimethylamino)butanoate. For example, the lipid formulation can beselected from the following:

LNP11 MC3/DSPC/Cholesterol/PEG-DMG 50/10/38.5/1.5 LNP14MC3/DSPC/Cholesterol/PEG-DMG 40/15/40/5 LNP15MC3/DSPC/Cholesterol/PEG-DSG/GalNAc-PEG-DSG 50/10/35/4.5/0.5 LNP16MC3/DSPC/Cholesterol/PEG-DMG 50/10/38.5/1.5 LNP17MC3/DSPC/Cholesterol/PEG-DSG 50/10/38.5/1.5 LNP18MC3/DSPC/Cholesterol/PEG-DMG 50/10/38.5/1.5 LNP19MC3/DSPC/Cholesterol/PEG-DMG 50/10/35/5 LNP20MC3/DSPC/Cholesterol/PEG-DPG 50/10/38.5/1.5

In other embodiments, the cationic lipid comprises formula A whereinformula A is

where R1 and R2 are independently alkyl, alkenyl or alkynyl, each can beoptionally substituted, and R3 and R4 are independently lower alkyl orR3 and R4 can be taken together to form an optionally substitutedheterocyclic ring. In some embodiments the cationic lipid comprisesformula A and is XTC(2,2-Dilinoleyl-4-dimethylaminoethyl[1,3]-dioxolane). The lipidformulation can include the cationic lipid XTC, the non-cationic lipidDSPC, the sterol cholesterol and the PEG lipid PEG-DMG. In otherembodiments the cationic lipid comprises XTC and the formulation isselected from the group consisting of:

LNP05 XTC/DSPC/Cholesterol/PEG-DMG 57.5/7.5/31.5/3.5 LNP06XTC/DSPC/Cholesterol/PEG-DMG 57.5/7.5/31.5/3.5 LNP07XTC/DSPC/Cholesterol/PEG-DMG 60/7.5/31/1.5, LNP08XTC/DSPC/Cholesterol/PEG-DMG 60/7.5/31/1.5 LNP09XTC/DSPC/Cholesterol/PEG-DMG 50/10/38.5/1.5 LNP13XTC/DSPC/Cholesterol/PEG-DMG 50/10/38.5/1.5 LNP22XTC/DSPC/Cholesterol/PEG-DSG 50/10/38.5/1.5

In still further embodiments, the cationic lipid comprises ALNY-100((3aR,5s,6aS)—N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine)).For example, the cationic lipid comprises ALNY-100 and the formulationconsists of ALNY-100/DSPC/Cholesterol/PEG-DMG in a ratio of50/10/38.5/1.5

The composition includes a dsRNA targeting PCSK9. In some embodiments,the sense strand comprises SEQ ID NO:1227 and the antisense strandcomprises SEQ ID NO:1228. In other embodiments, the sense strandconsists of SEQ ID NO:1227 and the antisense strand consists of SEQ IDNO:1228. One or both strands can be modified, e.g., each strand ismodified as follows to include a 2′-O-methyl ribonucleotide as indicatedby a lower case letter “c” or “u” and a phosphorothioate as indicated bya lower case letter “s”: the dsRNA consists of a sense strand consistingof

SEQ ID NO: 1229 (5′- uucuAGAccuGuuuuGcuuTsT -3′)

and an antisense strand consisting of

SEQ ID NO: 1230 (5′- AAGcAAAAcAGGUCuAGAATsT-3′).

In other embodiments, the dsRNA comprises at least one modifiednucleotide, e.g., a modified nucleotide chosen from the group of: a2′-O-methyl modified nucleotide, a nucleotide comprising a5′-phosphorothioate group, and a terminal nucleotide linked to acholesteryl derivative or dodecanoic acid bisdecylamide group, and/or,e.g., the modified nucleotide is chosen from the group of: a2′-deoxy-2′-fluoro modified nucleotide, a 2′-deoxy-modified nucleotide,a locked nucleotide, an abasic nucleotide, 2′-amino-modified nucleotide,2′-alkyl-modified nucleotide, morpholino nucleotide, a phosphoramidate,and a non-natural base comprising nucleotide. In one embodiment, dsRNAcomprises at least one 2′-O-methyl modified ribonucleotide and at leastone nucleotide comprising a 5′-phosphorothioate group.

The compositions include a dsRNA between 15 and 30 base pairs in length.In one embodiment, each strand of the dsRNA is 19-23 bases in length,or, e.g., 21-23 bases in length, or, e.g. 21 bases in length.

In one aspect, the compositions include a lipoprotein, e.g.,apolipoprotein E (ApoE). In some embodiments, the compositions include alipoprotein and the dsRNA is conjugated to a lipophile, e.g., acholesterol. The ApoE can be reconstituted with at least one amphiphilicagent, e.g., a phospholipid, e.g., a phospholipid selected from thegroup consisting of dimyristoyl phosphatidyl choline (DMPC),dioleoylphosphatidylethanolamine (DOPE),palmitoyloleoylphosphatidylcholine (POPC), egg phosphatidylcholine(EPC), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine(DOPC), dipalmitoylphosphatidylcholine (DPPC),dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol(DPPG), -phosphatidylethanolamine (POPE),dioleoyl-phosphatidylethanolamine4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), andcombinations thereof. In some embodiments, the ApoE is reconstitutedhigh density lipoprotein (rHDL).

The compositions, e.g., lipid formulated dsRNA targeting PCSK9, can beadministered to a cell or subject, e.g., a primate, e.g., a human. Inone aspect, administration of the compositions inhibits expression ofPCSK9 by at least 40% compared to administration of a control and/orreduces PCSK9 protein levels in the mammal compared to administration ofa control, and/or reduces LDLc levels in a mammal compared toadministration of a control and/or reduces both PCSK9 hepatic mRNA andtotal serum cholesterol at a dosage of less than 0.1 mg/kg compared toadministration of a control and/or reduces PCSK9 hepatic mRNA at an ED50of about 0.2 mg/kg and reduces total serum cholesterol with an ED50 ofabout 0.08 mg/kg compared to administration of a control and/or reducesserum LDL particle numbers by more than 90% or reduces serum HDLparticle numbers by more than 75% compared to administration of acontrol.

The invention also provides methods comprising administering the lipidformulated PCSK9 targeted dsRNA compositions described herein. In oneembodiment, the invention includes a method for inhibiting theexpression of PCSK9 in a cell comprising administering the compositionsto the cell. In another embodiment, the invention includes a method forreducing LDLc levels in a mammal in need of treatment comprisingadministering the compositions to the mammal.

As described in more detail below, the methods can include anyappropriate dosage, e.g., between 0.25 mg/kg and 4 mg/kg dsRNA, or e.g.,at about 0.01, 0.1, 0.5, 1.0, 2.5, or 5.0 mg/kg dsRNA.

Also described herein are methods for inhibiting expression of a PCSK9gene in a subject, comprising administering to the subject the lipidformulated PCSK9 targeted dsRNA compositions described herein at a firstdose of about 3 mg/kg followed by administering at least one subsequentdose once a week, wherein the subsequent dose is lower than the firstdose. The subject can be, .e.g., a rat or a non-human primate or ahuman. The subsequent dose can be about 1.0 mg/kg or about 0.3 mg/kg. Insome embodiments, the subsequent dose is administered once a week forfour weeks. In some embodiments, administration of the first dosedecreases total cholesterol levels by about 15-60%.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The prefixes “AD-” “DP-” and “AL-DP-” are used interchangeably e.g.,AL-DP-9327 and AD-9237.

FIG. 1 shows the structure of the ND-98 lipid.

FIG. 2 shows the results of the in vivo screen of 16 mouse specific(AL-DP-9327 through AL-DP-9342) PCSK9 siRNAs directed against differentORF regions of PCSK9 mRNA (having the first nucleotide corresponding tothe ORF position indicated on the graph) in C57/BL6 mice (5animals/group). The ratio of PCSK9 mRNA to GAPDH mRNA in liver lysateswas averaged over each treatment group and compared to a control grouptreated with PBS or a control group treated with an unrelated siRNA(blood coagulation factor VII).

FIG. 3 shows the results of the in vivo screen of 16 human/mouse/ratcross-reactive (AL-DP-9311 through AL-DP-9326) PCSK9 siRNAs directedagainst different ORF regions of PCSK9 mRNA (having the first nucleotidecorresponding to the ORF position indicated on the graph) in C57/BL6mice (5 animals/group). The ratio of PCSK9 mRNA to GAPDH mRNA in liverlysates was averaged over each treatment group and compared to a controlgroup treated with PBS or a control group treated with an unrelatedsiRNA (blood coagulation factor VII).

FIG. 4 shows the results of the in vivo screen of 16 mouse specific(AL-DP-9327 through AL-DP-9342) PCSK9 siRNAs in C57/BL6 mice (5animals/group). Total serum cholesterol levels were averaged over eachtreatment group and compared to a control group treated with PBS or acontrol group treated with an unrelated siRNA (blood coagulation factorVII).

FIG. 5 shows the results of the in vivo screen of 16 human/mouse/ratcross-reactive (AL-DP-9311 through AL-DP-9326) PCSK9 siRNAs in C57/BL6mice (5 animals/group). Total serum cholesterol levels were averagedover each treatment group and compared to a control group treated withPBS or a control group treated with an unrelated siRNA (bloodcoagulation factor VII).

FIGS. 6A and 6B compare in vitro and in vivo results, respectively, forsilencing PCSK9.

FIG. 7A and FIG. 7B are an example of in vitro results for silencingPCSK9 using monkey primary hepatocytes.

FIG. 7C show results for silencing of PCSK9 in monkey primaryhepatocytes using AL-DP-9680 and chemically modified version ofAL-DP-9680.

FIG. 8 shows in vivo activity of LNP-01 formulated siRNAs to PCSK-9.

FIGS. 9A and 9B show in vivo activity of LNP-01 Formulated chemicallymodified 9314 and derivatives with chemical modifications such asAD-10792, AD-12382, AD-12384, AD-12341 at different times post a singledose in mice.

FIG. 10A shows the effect of PCSK9 siRNAs on PCSK9 transcript levels andtotal serum cholesterol levels in rats after a single dose of formulatedAD-10792. FIG. 10B shows the effect of PCSK9 siRNAs on serum totalcholesterol levels in the experiment as 10A. A single dose of formulatedAD-10792 results in an ˜60% lowering of total cholesterol in the ratsthat returns to baseline by ˜3-4 weeks. FIG. 10C shows the effect ofPCSK9 siRNAs on hepatic cholesterol and triglyceride levels in the sameexperiment as 10A.

FIG. 11 is a Western blot showing that liver LDL receptor levels wereupregulated following administration of PCSK9 siRNAs in rat.

FIGS. 12A-12D show the effects of PCSK9 siRNAs on LDLc and ApoB proteinlevels, total cholesterol/HDLc ratios, and PCSK9 protein levels,respectively, in nonhuman primates following a single dose of formulatedAD-10792 or AD-9680.

FIG. 13A is a graph showing that unmodified siRNA-AD-A1A (AD-9314), butnot 2′OMe modified siRNA-AD-1A2 (AD-10792), induced IFN-alpha in humanprimary blood monocytes. FIG. 13B is a graph showing that unmodifiedsiRNA-AD-A1A (AD-9314), but not 2′OMe modified siRNA-AD-1A2 (AD-10792),also induced TNF-alpha in human primary blood monocytes.

FIG. 14A is a graph showing that the PCSK9 siRNA siRNA-AD-1A2 (a.k.a.LNP-PCS-A2 or a.k.a. “formulated AD-10792”) decreased PCSK9 mRNA levelsin mice liver in a dose-dependent manner. FIG. 14B is a graph showingthat single administration of 5 mg/kg siRNA-AD-1A2 decreased serum totalcholesterol levels in mice within 48 hours.

FIG. 15A is a graph showing that PCSK9 siRNAs targeting human and monkeyPCSK9 (LNP-PCS-C2) (a.k.a. “formulated AD-9736”), and PCSK9 siRNAstargeting mouse PCSK9 (LNP-PCS-A2) (a.k.a. “formulated AD-10792”),reduced liver PCSK9 levels in transgenic mice expressing human PCSK9.FIG. 15B is a graph showing that LNP-PCS-C2 and LNP-PCS-A2 reducedplasma PCSK9 levels in the same transgenic mice.

FIG. 16 shows the structure of an siRNA conjugated to Chol-p-(GalNAc)₃via phosphate linkage at the 3′ end.

FIG. 17 shows the structure of an siRNA conjugated to LCO(GalNAc)₃ (a(GalNAc)3-3′-Lithocholic-oleoyl siRNA Conjugate).

FIG. 18 is a graph showing the results of conjugated siRNAs on PCSK9transcript levels and total serum cholesterol in mice.

FIG. 19 is a graph showing the results of lipid formulated siRNAs onPCSK9 transcript levels and total serum cholesterol in rats.

FIG. 20 is a graph showing the results of siRNA transfection on PCSK9transcript levels in HeLa cells using AD-9680 and variations of AD-9680as described in Table 6.

FIG. 21 is a graph showing the results of siRNA transfection on PCSK9transcript levels in HeLa cells using AD-14676 and variations ofAD-14676 as described in Table 6.

FIG. 22 is a graph with the results of PCSK9 targeted siRNA transfectionof Hep3B cells and the effects on PCSK9 and off-target gene levels.

FIG. 23 shows the results of treatment in rats with a maintenance doseof PCSK9 targeted siRNA.

FIG. 24 is a dose response curve of treatment of HeLa cells withmodified siRNAs.

FIG. 25 is a graph of average IC50 of siRNA vs. target position in humanPCSK9 transcript. The large blue dot indicates the IC50 and location ofAD-9680.

FIG. 26 is a graph with the results of administration of rEHDLformulated cholesterol conjugated siRNA.

FIG. 27A is a graph with results of administration of second generationLNP formulated PCSK9 targeted siRNA (AD-9680 in LNP11) to non-humanprimates, demonstrating a reduction in both PCSK9 protein and LDLclevels. LDLc: low density lipoprotein cholesterol; mpk: mg per kg.

FIG. 27B is a bar graph showing dose dependent PCSK mRNA silencing innon-human primates after treatment with LNP formulated siRNA targetingPCSK9.

FIG. 27C is a graph with the results of administration of secondgeneration LNP formulated PCSK9 targeted siRNA (AD-9680) to non-humanprimates, demonstrating a no change in HDLc levels.

FIG. 28 illustrates the chemical structures of the cationic lipids MC3and ALNY-100.

FIG. 29 is a graph of effects on PCSK9 mRNA and serum cholesterol levelsin rats after administration of LNP-09 formulated AD-10792, an siRNAtargeting rodent PCSK9.

FIG. 30 are graphs of the effects on PCSK9 mRNA and LDL/HDL particlenumbers in CETP/ApoB tg mice after administration of LNP-09 formulatedAD-10792, an siRNA targeting rodent PCSK9.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides a solution to the problem of treating diseasesthat can be modulated by the down regulation of the PCSK9 gene, such ashyperlipidemia, by using double-stranded ribonucleic acid (dsRNA) tosilence the PCSK9 gene.

The invention provides compositions and methods for inhibiting theexpression of the PCSK9 gene in a subject using a dsRNA. The inventionalso provides compositions and methods for treating pathologicalconditions and diseases, such as hyperlipidemia, that can be modulatedby down regulating the expression of the PCSK9 gene. dsRNA directs thesequence-specific degradation of mRNA through a process known as RNAinterference (RNAi).

The dsRNA useful for the compositions and methods of an inventioninclude an RNA strand (the antisense strand) having a region that isless than 30 nucleotides in length, generally 19-24 nucleotides inlength, and is substantially complementary to at least part of an mRNAtranscript of the PCSK9 gene. The use of these dsRNAs enables thetargeted degradation of an mRNA that is involved in the regulation ofthe LDL Receptor and circulating cholesterol levels. Using cell-basedand animal assays, the present inventors have demonstrated that very lowdosages of these dsRNAs can specifically and efficiently mediate RNAi,resulting in significant inhibition of expression of the PCSK9 gene.Thus, methods and compositions including these dsRNAs are useful fortreating pathological processes that can be mediated by down regulatingPCSK9, such as in the treatment of hyperlipidemia.

The following detailed description discloses how to make and use thedsRNA and compositions containing dsRNA to inhibit the expression of thetarget PCSK9 gene, as well as compositions and methods for treatingdiseases that can be modulated by down regulating the expression ofPCSK9, such as hyperlipidemia. The pharmaceutical compositions of theinvention include a dsRNA having an antisense strand having a region ofcomplementarity that is less than 30 nucleotides in length, generally19-24 nucleotides in length, and that is substantially complementary toat least part of an RNA transcript of the PCSK9 gene, together with apharmaceutically acceptable carrier.

Accordingly, certain aspects of the invention provide pharmaceuticalcompositions including the dsRNA that targets PCSK9 together with apharmaceutically acceptable carrier, methods of using the compositionsto inhibit expression of the PCSK9 gene, and methods of using thepharmaceutical compositions to treat diseases by down regulating theexpression of PCSK9.

DEFINITIONS

For convenience, the meaning of certain terms and phrases used in thespecification, examples, and appended claims, are provided below. Ifthere is an apparent discrepancy between the usage of a term in otherparts of this specification and its definition provided in this section,the definition in this section shall prevail.

“G,” “C,” “A” and “U” each generally stand for a nucleotide thatcontains guanine, cytosine, adenine, and uracil as a base, respectively.“T” and “dT” are used interchangeably herein and refer to adeoxyribonucleotide wherein the nucleobase is thymine, e.g.,deoxyribothymine. However, it will be understood that the term“ribonucleotide” or “nucleotide” or “deoxyribonucleotide” can also referto a modified nucleotide, as further detailed below, or a surrogatereplacement moiety. The skilled person is well aware that guanine,cytosine, adenine, and uracil may be replaced by other moieties withoutsubstantially altering the base pairing properties of an oligonucleotidecomprising a nucleotide bearing such replacement moiety. For example,without limitation, a nucleotide comprising inosine as its base may basepair with nucleotides containing adenine, cytosine, or uracil. Hence,nucleotides containing uracil, guanine, or adenine may be replaced inthe nucleotide sequences of the invention by a nucleotide containing,for example, inosine. Sequences comprising such replacement moieties areembodiments of the invention.

As used herein, “PCSK9” refers to the proprotein convertase subtilisinkexin 9 gene or protein (also known as FH3, HCHOLA3, NARC-1, NARC1).Examples of mRNA sequences to PCSK9 include but are not limited to thefollowing: human: NM_(—)174936; mouse: NM_(—)153565, and rat:NM_(—)199253. Additional examples of PCSK9 mRNA sequences are readilyavailable using, e.g., GenBank.

As used herein, “target sequence” refers to a contiguous portion of thenucleotide sequence of an mRNA molecule formed during the transcriptionof the PCSK9 gene, including mRNA that is a product of RNA processing ofa primary transcription product.

As used herein, the term “strand comprising a sequence” refers to anoligonucleotide comprising a chain of nucleotides that is described bythe sequence referred to using the standard nucleotide nomenclature.

As used herein, and unless otherwise indicated, the term“complementary,” when used to describe a first nucleotide sequence inrelation to a second nucleotide sequence, refers to the ability of anoligonucleotide or polynucleotide comprising the first nucleotidesequence to hybridize and form a duplex structure under certainconditions with an oligonucleotide or polynucleotide comprising thesecond nucleotide sequence, as will be understood by the skilled person.Such conditions can, for example, be stringent conditions, wherestringent conditions may include: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mMEDTA, 50° C. or 70° C. for 12-16 hours followed by washing. Otherconditions, such as physiologically relevant conditions as may beencountered inside an organism, can apply. The skilled person will beable to determine the set of conditions most appropriate for a test ofcomplementarity of two sequences in accordance with the ultimateapplication of the hybridized nucleotides.

This includes base-pairing of the oligonucleotide or polynucleotidehaving the first nucleotide sequence to the oligonucleotide orpolynucleotide having the second nucleotide sequence over the entirelength of the first and second nucleotide sequences. Such sequences canbe referred to as “fully complementary” with respect to each other.However, where a first sequence is referred to as “substantiallycomplementary” with respect to a second sequence herein, the twosequences can be fully complementary, or they may form one or more, butgenerally not more than 4, 3 or 2 mismatched base pairs uponhybridization, while retaining the ability to hybridize under theconditions most relevant to their ultimate application. However, wheretwo oligonucleotides are designed to form, upon hybridization, one ormore single stranded overhangs, such overhangs shall not be regarded asmismatches with regard to the determination of complementarity. Forexample, a dsRNA having one oligonucleotide 21 nucleotides in length andanother oligonucleotide 23 nucleotides in length, wherein the longeroligonucleotide has a sequence of 21 nucleotides that is fullycomplementary to the shorter oligonucleotide, may yet be referred to as“fully complementary.”

“Complementary” sequences, as used herein, may also include, or beformed entirely from, non-Watson-Crick base pairs and/or base pairsformed from non-natural and modified nucleotides, in as far as the aboverequirements with respect to their ability to hybridize are fulfilled.Such non-Watson-Crick base pairs includes, but not limited to, G:UWobble or Hoogstein base pairing.

The terms “complementary”, “fully complementary” and “substantiallycomplementary” herein may be used with respect to the base matchingbetween the sense strand and the antisense strand of a dsRNA, or betweenthe antisense strand of a dsRNA and a target sequence, as will beunderstood from the context of their use.

As used herein, a polynucleotide which is “substantially complementaryto at least part of” a messenger RNA (mRNA) refers to a polynucleotidethat is substantially complementary to a contiguous portion of the mRNAof interest (e.g., encoding PCSK9) including a 5′ UTR, an open readingframe (ORF), or a 3′ UTR. For example, a polynucleotide is complementaryto at least a part of a PCSK9 mRNA if the sequence is substantiallycomplementary to a non-interrupted portion of an mRNA encoding PCSK9.

The term “double-stranded RNA” or “dsRNA”, as used herein, refers aduplex structure comprising two anti-parallel and substantiallycomplementary, as defined above, nucleic acid strands. In general, themajority of nucleotides of each strand are ribonucleotides, but asdescribed in detail herein, each or both strands can also include atleast one non-ribonucleotide, e.g., a deoxyribonucleotide and/or amodified nucleotide. In addition, as used in this specification, “dsRNA”may include chemical modifications to ribonucleotides, includingsubstantial modifications at multiple nucleotides and including alltypes of modifications disclosed herein or known in the art. Any suchmodifications, as used in an siRNA type molecule, are encompassed by“dsRNA” for the purposes of this specification and claims.

The two strands forming the duplex structure may be different portionsof one larger RNA molecule, or they may be separate RNA molecules. Whereseparate RNA molecules, such dsRNA are often referred to in theliterature as siRNA (“short interfering RNA”). Where the two strands arepart of one larger molecule, and therefore are connected by anuninterrupted chain of nucleotides between the 3′-end of one strand andthe 5′ end of the respective other strand forming the duplex structure,the connecting RNA chain is referred to as a “hairpin loop”, “shorthairpin RNA” or “shRNA”. Where the two strands are connected covalentlyby means other than an uninterrupted chain of nucleotides between the3′-end of one strand and the 5′ end of the respective other strandforming the duplex structure, the connecting structure is referred to asa “linker”. The RNA strands may have the same or a different number ofnucleotides. The maximum number of base pairs is the number ofnucleotides in the shortest strand of the dsRNA minus any overhangs thatare present in the duplex. In addition to the duplex structure, a dsRNAmay comprise one or more nucleotide overhangs. In general, the majorityof nucleotides of each strand are ribonucleotides, but as described indetail herein, each or both strands can also include at least onenon-ribonucleotide, e.g., a deoxyribonucleotide and/or a modifiednucleotide. In addition, as used in this specification, “dsRNA” mayinclude chemical modifications to ribonucleotides, including substantialmodifications at multiple nucleotides and including all types ofmodifications disclosed herein or known in the art. Any suchmodifications, as used in an siRNA type molecule, are encompassed by“dsRNA” for the purposes of this specification and claims.

As used herein, a “nucleotide overhang” refers to the unpairednucleotide or nucleotides that protrude from the duplex structure of adsRNA when a 3′-end of one strand of the dsRNA extends beyond the 5′-endof the other strand, or vice versa. “Blunt” or “blunt end” means thatthere are no unpaired nucleotides at that end of the dsRNA, i.e., nonucleotide overhang. A “blunt ended” dsRNA is a dsRNA that isdouble-stranded over its entire length, i.e., no nucleotide overhang ateither end of the molecule. For clarity, chemical caps or non-nucleotidechemical moieties conjugated to the 3′ end or 5′ end of an siRNA are notconsidered in determining whether an siRNA has an overhang or is bluntended.

The term “antisense strand” refers to the strand of a dsRNA whichincludes a region that is substantially complementary to a targetsequence. As used herein, the term “region of complementarity” refers tothe region on the antisense strand that is substantially complementaryto a sequence, for example a target sequence, as defined herein. Wherethe region of complementarity is not fully complementary to the targetsequence, the mismatches may be in the internal or terminal regions ofthe molecule. Generally the most tolerated mismatches are in theterminal regions, e.g., within 6, 5, 4, 3, or 2 nucleotides of the 5′and/or 3′ terminus.

The term “sense strand,” as used herein, refers to the strand of a dsRNAthat includes a region that is substantially complementary to a regionof the antisense strand.

“Introducing into a cell”, when referring to a dsRNA, means facilitatinguptake or absorption into the cell, as is understood by those skilled inthe art. Absorption or uptake of dsRNA can occur through unaideddiffusive or active cellular processes, or by auxiliary agents ordevices. The meaning of this term is not limited to cells in vitro; adsRNA may also be “introduced into a cell”, wherein the cell is part ofa living organism. In such instance, introduction into the cell willinclude the delivery to the organism. For example, for in vivo delivery,dsRNA can be injected into a tissue site or administered systemically.In vitro introduction into a cell includes methods known in the art suchas electroporation and lipofection.

The terms “silence,” “inhibit the expression of,” “down-regulate theexpression of,” “suppress the expression of,” and the like, in as far asthey refer to the PCSK9 gene, herein refer to the at least partialsuppression of the expression of the PCSK9 gene, as manifested by areduction of the amount of PCSK9 mRNA which may be isolated from a firstcell or group of cells in which the PCSK9 gene is transcribed and whichhas or have been treated such that the expression of the PCSK9 gene isinhibited, as compared to a second cell or group of cells substantiallyidentical to the first cell or group of cells but which has or have notbeen so treated (control cells). The degree of inhibition is usuallyexpressed in terms of

${\frac{( {{mRNA}\mspace{14mu} {in}\mspace{14mu} {control}\mspace{14mu} {cells}} ) - ( {{mRNA}\mspace{14mu} {in}\mspace{14mu} {treated}\mspace{14mu} {cells}} )}{( {{mRNA}\mspace{14mu} {in}\mspace{14mu} {control}\mspace{14mu} {cells}} )} \cdot 100}\%$

Alternatively, the degree of inhibition may be given in terms of areduction of a parameter that is functionally linked to PCSK9 geneexpression, e.g. the amount of protein encoded by the PCSK9 gene whichis produced by a cell, or the number of cells displaying a certainphenotype. In principle, target gene silencing can be determined in anycell expressing the target, either constitutively or by genomicengineering, and by any appropriate assay. However, when a reference isneeded in order to determine whether a given dsRNA inhibits theexpression of the PCSK9 gene by a certain degree and therefore isencompassed by the instant invention, the assays provided in theExamples below shall serve as such reference.

As used herein in the context of PCSK9 expression, the terms “treat”,“treatment”, and the like, refer to relief from or alleviation ofpathological processes which can be mediated by down regulating thePCSK9 gene. In the context of the present invention insofar as itrelates to any of the other conditions recited herein below (other thanpathological processes which can be mediated by down regulating thePCSK9 gene), the terms “treat”, “treatment”, and the like mean torelieve or alleviate at least one symptom associated with suchcondition, or to slow or reverse the progression of such condition. Forexample, in the context of hyperlipidemia, treatment will involve adecrease in serum lipid levels.

As used herein, the phrases “therapeutically effective amount” and“prophylactically effective amount” refer to an amount that provides atherapeutic benefit in the treatment, prevention, or management ofpathological processes that can be mediated by down regulating the PCSK9gene or an overt symptom of pathological processes which can be mediatedby down regulating the PCSK9 gene. The specific amount that istherapeutically effective can be readily determined by an ordinarymedical practitioner, and may vary depending on factors known in theart, such as, e.g., the type of pathological processes that can bemediated by down regulating the PCSK9 gene, the patient's history andage, the stage of pathological processes that can be mediated by downregulating PCSK9 gene expression, and the administration of otheranti-pathological processes that can be mediated by down regulatingPCSK9 gene expression.

As used herein, a “pharmaceutical composition” includes apharmacologically effective amount of a dsRNA and a pharmaceuticallyacceptable carrier. As used herein, “pharmacologically effectiveamount,” “therapeutically effective amount” or simply “effective amount”refers to that amount of an RNA effective to produce the intendedpharmacological, therapeutic or preventive result. For example, if agiven clinical treatment is considered effective when there is at leasta 25% reduction in a measurable parameter associated with a disease ordisorder, a therapeutically effective amount of a drug for the treatmentof that disease or disorder is the amount necessary to effect at least a25% reduction in that parameter.

The term “pharmaceutically acceptable carrier” refers to a carrier foradministration of a therapeutic agent. Such carriers include, but arenot limited to, saline, buffered saline, dextrose, water, glycerol,ethanol, and combinations thereof and are described in more detailbelow. The term specifically excludes cell culture medium.

As used herein, a “transformed cell” is a cell into which a vector hasbeen introduced from which a dsRNA molecule may be expressed.

Double-Stranded Ribonucleic Acid (dsRNA)

As described in more detail below, the invention provides methods andcomposition having double-stranded ribonucleic acid (dsRNA) moleculesfor inhibiting the expression of the PCSK9 gene in a cell or mammal,wherein the dsRNA includes an antisense strand having a region ofcomplementarity that is complementary to at least a part of an mRNAformed in the expression of the PCSK9 gene, and wherein the region ofcomplementarity is less than 30 nucleotides in length, generally 19-24nucleotides in length. In some embodiments, the dsRNA, upon contact witha cell expressing the PCSK9 gene, inhibits the expression of said PCSK9gene, e.g., as measured such as by an assay described herein. Forexample, expression of a PCSK9 gene in cell culture, such as in HepB3cells, can be assayed by measuring PCSK9 mRNA levels, such as by bDNA orTaqMan assay, or by measuring protein levels, such as by ELISA assay.The dsRNA of the invention can further include one or moresingle-stranded nucleotide overhangs.

The dsRNA can be synthesized by standard methods known in the art asfurther discussed below, e.g., by use of an automated DNA synthesizer,such as are commercially available from, for example, Biosearch, AppliedBiosystems, Inc. The dsRNA includes two nucleic acid strands that aresufficiently complementary to hybridize to form a duplex structure. Onestrand of the dsRNA (the antisense strand) can have a region ofcomplementarity that is substantially complementary, and generally fullycomplementary, to a target sequence, derived from the sequence of anmRNA formed during the expression of the PCSK9 gene. The other strand(the sense strand) includes a region that is complementary to theantisense strand, such that the two strands hybridize and form a duplexstructure when combined under suitable conditions. Generally, the duplexstructure is between 15 and 30, or between 25 and 30, or between 18 and25, or between 19 and 24, or between 19 and 21, or 19, 20, or 21 basepairs in length. In one embodiment the duplex is 19 base pairs inlength. In another embodiment the duplex is 21 base pairs in length.When two different siRNAs are used in combination, the duplex lengthscan be identical or can differ.

Each strand of the dsRNA of invention is generally between 15 and 30, orbetween 18 and 25, or 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides inlength. In other embodiments, each is strand is 25-30 nucleotides inlength. Each strand of the duplex can be the same length or of differentlengths. When two different siRNAs are used in combination, the lengthsof each strand of each siRNA can be identical or can differ.

The dsRNA of the invention can include one or more single-strandedoverhang(s) of one or more nucleotides. In one embodiment, at least oneend of the dsRNA has a single-stranded nucleotide overhang of 1 to 4,generally 1 or 2 nucleotides. In another embodiment, the antisensestrand of the dsRNA has 1-10 nucleotides overhangs each at the 3′ endand the 5′ end over the sense strand. In further embodiments, the sensestrand of the dsRNA has 1-10 nucleotides overhangs each at the 3′ endand the 5′ end over the antisense strand.

A dsRNAs having at least one nucleotide overhang can have unexpectedlysuperior inhibitory properties than the blunt-ended counterpart. In someembodiments the presence of only one nucleotide overhang strengthens theinterference activity of the dsRNA, without affecting its overallstability. A dsRNA having only one overhang has proven particularlystable and effective in vivo, as well as in a variety of cells, cellculture mediums, blood, and serum. Generally, the single-strandedoverhang is located at the 3′-terminal end of the antisense strand or,alternatively, at the 3′-terminal end of the sense strand. The dsRNA canalso have a blunt end, generally located at the 5′-end of the antisensestrand. Such dsRNAs can have improved stability and inhibitory activity,thus allowing administration at low dosages, i.e., less than 5 mg/kgbody weight of the recipient per day. Generally, the antisense strand ofthe dsRNA has a nucleotide overhang at the 3′-end, and the 5′-end isblunt. In another embodiment, one or more of the nucleotides in theoverhang is replaced with a nucleoside thiophosphate.

The dsRNA can be synthesized by standard methods known in the art asfurther discussed below, e.g., by use of an automated DNA synthesizer,such as are commercially available from, for example, Biosearch, AppliedBiosystems, Inc. In one embodiment, the PCSK9 gene is a human PCSK9gene. In other embodiments, the antisense strand of the dsRNA includes afirst strand selected from the sense sequences of Table 1a, Table 2a,and Table 5a, and a second strand selected from the antisense sequencesof Table 1a, Table 2a, and Table 5a. Alternative antisense agents thattarget elsewhere in the target sequence provided in Table 1a, Table 2a,and Table 5a, can readily be determined using the target sequence andthe flanking PCSK9 sequence.

For example, the dsRNA AD-9680 (from Table 1a) targets the PCSK 9 geneat 3530-3548; therefore the target sequence is as follows: 5′UUCUAGACCUGUUUUGCUU 3′ (SEQ ID NO:1523). The dsRNA AD-10792 (from Table1a) targets the PCSK9 gene at 1091-1109; therefore the target sequenceis as follows: 5′ GCCUGGAGUUUAUUCGGAA 3′ (SEQ ID NO:1524). Included inthe invention are dsRNAs that have regions of complementarity to SEQ IDNO:1523 and SEQ ID NO:1524.

In further embodiments, the dsRNA includes at least one nucleotidesequence selected from the groups of sequences provided in Table 1a,Table 2a, and Table 5a. In other embodiments, the dsRNA includes atleast two sequences selected from this group, where one of the at leasttwo sequences is complementary to another of the at least two sequences,and one of the at least two sequences is substantially complementary toa sequence of an mRNA generated in the expression of the PCSK9 gene.Generally, the dsRNA includes two oligonucleotides, where oneoligonucleotide is described as the sense strand in Table 1a, Table 2a,and Table 5a and the second oligonucleotide is described as theantisense strand in Table 1a, Table 2a, and Table 5a

The skilled person is well aware that dsRNAs having a duplex structureof between 20 and 23, but specifically 21, base pairs have been hailedas particularly effective in inducing RNA interference (Elbashir et al.,EMBO 2001, 20:6877-6888). However, others have found that shorter orlonger dsRNAs can be effective as well. In the embodiments describedabove, by virtue of the nature of the oligonucleotide sequences providedin Table 1a, Table 2a, and Table 5a, the dsRNAs of the invention caninclude at least one strand of a length of minimally 21 nt. It can bereasonably expected that shorter dsRNAs having one of the sequences ofTable 1a, Table 2a, and Table 5a minus only a few nucleotides on one orboth ends may be similarly effective as compared to the dsRNAs describedabove. Hence, dsRNAs having a partial sequence of at least 15, 16, 17,18, 19, 20, or more contiguous nucleotides from one of the sequences ofTable 1a, Table 2a, and Table 5a, and differing in their ability toinhibit the expression of the PCSK9 gene in a FACS assay as describedherein below by not more than 5, 10, 15, 20, 25, or 30% inhibition froma dsRNA comprising the full sequence, are contemplated by the invention.Further dsRNAs that cleave within the target sequence provided in Table1a, Table 2a, and Table 5a can readily be made using the PCSK9 sequenceand the target sequence provided.

In addition, the RNAi agents provided in Table 1a, Table 2a, and Table5a identify a site in the PCSK9 mRNA that is susceptible to RNAi basedcleavage. As such the present invention further includes RNAi agentsthat target within the sequence targeted by one of the agents of thepresent invention. As used herein a second RNAi agent is said to targetwithin the sequence of a first RNAi agent if the second RNAi agentcleaves the message anywhere within the mRNA that is complementary tothe antisense strand of the first RNAi agent. Such a second agent willgenerally consist of at least 15 contiguous nucleotides from one of thesequences provided in Table 1a, Table 2a, and Table 5a coupled toadditional nucleotide sequences taken from the region contiguous to theselected sequence in the PCSK9 gene. For example, the last 15nucleotides of SEQ ID NO:1 (minus the added AA sequences) combined withthe next 6 nucleotides from the target PCSK9 gene produces a singlestrand agent of 21 nucleotides that is based on one of the sequencesprovided in Table 1a, Table 2a, and Table 5a.

The dsRNA of the invention can contain one or more mismatches to thetarget sequence. In one embodiment, the dsRNA of the invention containsno more than 1, no more than 2, or no more than 3 mismatches. In oneembodiment, the antisense strand of the dsRNA contains mismatches to thetarget sequence, and the area of mismatch is not located in the centerof the region of complementarity. In another embodiment, the antisensestrand of the dsRNA contains mismatches to the target sequence and themismatch is restricted to 5 nucleotides from either end, for example 5,4, 3, 2, or 1 nucleotide from either the 5′ or 3′ end of the region ofcomplementarity. For example, for a 23 nucleotide dsRNA strand which iscomplementary to a region of the PCSK9 gene, the dsRNA does not containany mismatch within the central 13 nucleotides. The methods describedwithin the invention can be used to determine whether a dsRNA containinga mismatch to a target sequence is effective in inhibiting theexpression of the PCSK9 gene. Consideration of the efficacy of dsRNAswith mismatches in inhibiting expression of the PCSK9 gene is important,especially if the particular region of complementarity in the PCSK9 geneis known to have polymorphic sequence variation within the population.

In one embodiment, at least one end of the dsRNA has a single-strandednucleotide overhang of 1 to 4, generally 1 or 2 nucleotides. dsRNAshaving at least one nucleotide overhang have unexpectedly superiorinhibitory properties than their blunt-ended counterparts. Moreover, thepresent inventors have discovered that the presence of only onenucleotide overhang strengthens the interference activity of the dsRNA,without affecting its overall stability. dsRNA having only one overhanghas proven particularly stable and effective in vivo, as well as in avariety of cells, cell culture mediums, blood, and serum. Generally, thesingle-stranded overhang is located at the 3′-terminal end of theantisense strand or, alternatively, at the 3′-terminal end of the sensestrand. The dsRNA may also have a blunt end, generally located at the5′-end of the antisense strand. Such dsRNAs have improved stability andinhibitory activity, thus allowing administration at low dosages, i.e.,less than 5 mg/kg body weight of the recipient per day. Generally, theantisense strand of the dsRNA has a nucleotide overhang at the 3′-end,and the 5′-end is blunt. In another embodiment, one or more of thenucleotides in the overhang is replaced with a nucleoside thiophosphate.

Chemical Modifications and Conjugates

In yet another embodiment, the dsRNA is chemically modified to enhancestability. The nucleic acids of the invention may be synthesized and/ormodified by methods well established in the art, such as those describedin “Current protocols in nucleic acid chemistry”, Beaucage, S. L. et al.(Edrs.), John Wiley & Sons, Inc., New York, N.Y., USA, which is herebyincorporated herein by reference. Chemical modifications may include,but are not limited to 2′ modifications, modifications at other sites ofthe sugar or base of an oligonucleotide, introduction of non-naturalbases into the oligonucleotide chain, covalent attachment to a ligand orchemical moiety, and replacement of internucleotide phosphate linkageswith alternate linkages such as thiophosphates. More than one suchmodification may be employed.

Chemical linking of the two separate dsRNA strands may be achieved byany of a variety of well-known techniques, for example by introducingcovalent, ionic or hydrogen bonds; hydrophobic interactions, van derWaals or stacking interactions; by means of metal-ion coordination, orthrough use of purine analogues. Generally, the chemical groups that canbe used to modify the dsRNA include, without limitation, methylene blue;bifunctional groups, generally bis-(2-chloroethyl)amine;N-acetyl-N′-(p-glyoxylbenzoyl)cystamine; 4-thiouracil; and psoralen. Inone embodiment, the linker is a hexa-ethylene glycol linker. In thiscase, the dsRNA are produced by solid phase synthesis and thehexa-ethylene glycol linker is incorporated according to standardmethods (e.g., Williams, D. J., and K. B. Hall, Biochem. (1996)35:14665-14670). In a particular embodiment, the 5′-end of the antisensestrand and the 3′-end of the sense strand are chemically linked via ahexaethylene glycol linker. In another embodiment, at least onenucleotide of the dsRNA comprises a phosphorothioate orphosphorodithioate groups. The chemical bond at the ends of the dsRNA isgenerally formed by triple-helix bonds. Table 1a, Table 2a, and Table 5aprovides examples of modified RNAi agents of the invention.

In yet another embodiment, the nucleotides at one or both of the twosingle strands may be modified to prevent or inhibit the degradationactivities of cellular enzymes, such as, for example, withoutlimitation, certain nucleases. Techniques for inhibiting the degradationactivity of cellular enzymes against nucleic acids are known in the artincluding, but not limited to, 2′-amino modifications, 2′-amino sugarmodifications, 2′-F sugar modifications, 2′-F modifications, 2′-alkylsugar modifications, uncharged backbone modifications, morpholinomodifications, 2′-O-methyl modifications, and phosphoramidate (see,e.g., Wagner, Nat. Med. (1995) 1:1116-8). Thus, at least one 2′-hydroxylgroup of the nucleotides on a dsRNA is replaced by a chemical group,generally by a 2′-amino or a 2′-methyl group. Also, at least onenucleotide may be modified to form a locked nucleotide. Such lockednucleotide contains a methylene bridge that connects the 2′-oxygen ofribose with the 4′-carbon of ribose. Oligonucleotides containing thelocked nucleotide are described in Koshkin, A. A., et al., Tetrahedron(1998), 54: 3607-3630) and Obika, S. et al., Tetrahedron Lett. (1998),39: 5401-5404). Introduction of a locked nucleotide into anoligonucleotide improves the affinity for complementary sequences andincreases the melting temperature by several degrees (Braasch, D. A. andD. R. Corey, Chem. Biol. (2001), 8:1-7).

Conjugating a ligand to a dsRNA can enhance its cellular absorption aswell as targeting to a particular tissue or uptake by specific types ofcells such as liver cells. In certain instances, a hydrophobic ligand isconjugated to the dsRNA to facilitate direct permeation of the cellularmembrane and or uptake across the liver cells. Alternatively, the ligandconjugated to the dsRNA is a substrate for receptor-mediatedendocytosis. These approaches have been used to facilitate cellpermeation of antisense oligonucleotides as well as dsRNA agents. Forexample, cholesterol has been conjugated to various antisenseoligonucleotides resulting in compounds that are substantially moreactive compared to their non-conjugated analogs. See M. ManoharanAntisense & Nucleic Acid Drug Development 2002, 12, 103. Otherlipophilic compounds that have been conjugated to oligonucleotidesinclude 1-pyrene butyric acid, 1,3-bis-O-(hexadecyl)glycerol, andmenthol. One example of a ligand for receptor-mediated endocytosis isfolic acid. Folic acid enters the cell by folate-receptor-mediatedendocytosis. dsRNA compounds bearing folic acid would be efficientlytransported into the cell via the folate-receptor-mediated endocytosis.Li and coworkers report that attachment of folic acid to the 3′-terminusof an oligonucleotide resulted in an 8-fold increase in cellular uptakeof the oligonucleotide. Li, S.; Deshmukh, H. M.; Huang, L. Pharm. Res.1998, 15, 1540. Other ligands that have been conjugated tooligonucleotides include polyethylene glycols, carbohydrate clusters,cross-linking agents, porphyrin conjugates, delivery peptides and lipidssuch as cholesterol and cholesterylamine. Examples of carbohydrateclusters include Chol-p-(GalNAc)₃ (N-acetyl galactosamine cholesterol)and LCO(GalNAc)₃ (N-acetyl galactosamine-3′-Lithocholic-oleoyl.

In certain instances, conjugation of a cationic ligand tooligonucleotides results in improved resistance to nucleases.Representative examples of cationic ligands are propylammonium anddimethylpropylammonium. Interestingly, antisense oligonucleotides werereported to retain their high binding affinity to mRNA when the cationicligand was dispersed throughout the oligonucleotide. See M. ManoharanAntisense & Nucleic Acid Drug Development 2002, 12, 103 and referencestherein.

In some cases, a ligand can be multifunctional and/or a dsRNA can beconjugated to more than one ligand. For example, the dsRNA can beconjugated to one ligand for improved uptake and to a second ligand forimproved release.

The ligand-conjugated dsRNA of the invention may be synthesized by theuse of a dsRNA that bears a pendant reactive functionality, such as thatderived from the attachment of a linking molecule onto the dsRNA. Thisreactive oligonucleotide may be reacted directly withcommercially-available ligands, ligands that are synthesized bearing anyof a variety of protecting groups, or ligands that have a linking moietyattached thereto. The methods of the invention facilitate the synthesisof ligand-conjugated dsRNA by the use of, in some embodiments,nucleoside monomers that have been appropriately conjugated with ligandsand that may further be attached to a solid-support material. Suchligand-nucleoside conjugates, optionally attached to a solid-supportmaterial, are prepared according to certain embodiments of the methodsdescribed herein via reaction of a selected serum-binding ligand with alinking moiety located on the 5′ position of a nucleoside oroligonucleotide. In certain instances, a dsRNA bearing an aralkyl ligandattached to the 3′-terminus of the dsRNA is prepared by first covalentlyattaching a monomer building block to a controlled-pore-glass supportvia a long-chain aminoalkyl group. Then, nucleotides are bonded viastandard solid-phase synthesis techniques to the monomer building-blockbound to the solid support. The monomer building block may be anucleoside or other organic compound that is compatible with solid-phasesynthesis.

The dsRNA used in the conjugates of the invention may be convenientlyand routinely made through the well-known technique of solid-phasesynthesis. Equipment for such synthesis is sold by several vendorsincluding, for example, Applied Biosystems (Foster City, Calif.). Anyother means for such synthesis known in the art may additionally oralternatively be employed. It is also known to use similar techniques toprepare other oligonucleotides, such as the phosphorothioates andalkylated derivatives.

Synthesis

Teachings regarding the synthesis of particular modifiedoligonucleotides may be found in the following U.S. patents: U.S. Pat.Nos. 5,138,045 and 5,218,105, drawn to polyamine conjugatedoligonucleotides; U.S. Pat. No. 5,212,295, drawn to monomers for thepreparation of oligonucleotides having chiral phosphorus linkages; U.S.Pat. Nos. 5,378,825 and 5,541,307, drawn to oligonucleotides havingmodified backbones; U.S. Pat. No. 5,386,023, drawn to backbone-modifiedoligonucleotides and the preparation thereof through reductive coupling;U.S. Pat. No. 5,457,191, drawn to modified nucleobases based on the3-deazapurine ring system and methods of synthesis thereof; U.S. Pat.No. 5,459,255, drawn to modified nucleobases based on N-2 substitutedpurines; U.S. Pat. No. 5,521,302, drawn to processes for preparingoligonucleotides having chiral phosphorus linkages; U.S. Pat. No.5,539,082, drawn to peptide nucleic acids; U.S. Pat. No. 5,554,746,drawn to oligonucleotides having β-lactam backbones; U.S. Pat. No.5,571,902, drawn to methods and materials for the synthesis ofoligonucleotides; U.S. Pat. No. 5,578,718, drawn to nucleosides havingalkylthio groups, wherein such groups may be used as linkers to othermoieties attached at any of a variety of positions of the nucleoside;U.S. Pat. Nos. 5,587,361 and 5,599,797, drawn to oligonucleotides havingphosphorothioate linkages of high chiral purity; U.S. Pat. No.5,506,351, drawn to processes for the preparation of 2′-O-alkylguanosine and related compounds, including 2,6-diaminopurine compounds;U.S. Pat. No. 5,587,469, drawn to oligonucleotides having N-2substituted purines; U.S. Pat. No. 5,587,470, drawn to oligonucleotideshaving 3-deazapurines; U.S. Pat. No. 5,223,168, and U.S. Pat. No.5,608,046, both drawn to conjugated 4′-desmethyl nucleoside analogs;U.S. Pat. Nos. 5,602,240, and 5,610,289, drawn to backbone-modifiedoligonucleotide analogs; U.S. Pat. Nos. 6,262,241, and 5,459,255, drawnto, inter alia, methods of synthesizing 2′-fluoro-oligonucleotides.

In the ligand-conjugated dsRNA and ligand-molecule bearingsequence-specific linked nucleosides of the invention, theoligonucleotides and oligonucleosides may be assembled on a suitable DNAsynthesizer utilizing standard nucleotide or nucleoside precursors, ornucleotide or nucleoside conjugate precursors that already bear thelinking moiety, ligand-nucleotide or nucleoside-conjugate precursorsthat already bear the ligand molecule, or non-nucleoside ligand-bearingbuilding blocks.

When using nucleotide-conjugate precursors that already bear a linkingmoiety, the synthesis of the sequence-specific linked nucleosides istypically completed, and the ligand molecule is then reacted with thelinking moiety to form the ligand-conjugated oligonucleotide.Oligonucleotide conjugates bearing a variety of molecules such assteroids, vitamins, lipids and reporter molecules, has previously beendescribed (see Manoharan et al., PCT Application WO 93/07883). In oneembodiment, the oligonucleotides or linked nucleosides featured in theinvention are synthesized by an automated synthesizer usingphosphoramidites derived from ligand-nucleoside conjugates in additionto the standard phosphoramidites and non-standard phosphoramidites thatare commercially available and routinely used in oligonucleotidesynthesis.

The incorporation of a 2′-O-methyl, 2′-O-ethyl, 2′-O-propyl, 2′-O-allyl,2′-β-aminoalkyl or 2′-deoxy-2′-fluoro group in nucleosides of anoligonucleotide confers enhanced hybridization properties to theoligonucleotide. Further, oligonucleotides containing phosphorothioatebackbones have enhanced nuclease stability. Thus, functionalized, linkednucleosides of the invention can be augmented to include either or botha phosphorothioate backbone or a 2′-O-methyl, 2′-O-ethyl, 2′-O-propyl,2′-β-aminoalkyl, 2′-O-allyl or 2′-deoxy-2′-fluoro group. A summarylisting of some of the oligonucleotide modifications known in the art isfound at, for example, PCT Publication WO 200370918.

In some embodiments, functionalized nucleoside sequences of theinvention possessing an amino group at the 5′-terminus are preparedusing a DNA synthesizer, and then reacted with an active esterderivative of a selected ligand. Active ester derivatives are well knownto those skilled in the art. Representative active esters includeN-hydrosuccinimide esters, tetrafluorophenolic esters,pentafluorophenolic esters and pentachlorophenolic esters. The reactionof the amino group and the active ester produces an oligonucleotide inwhich the selected ligand is attached to the 5′-position through alinking group. The amino group at the 5′-terminus can be preparedutilizing a 5′-Amino-Modifier C6 reagent. In one embodiment, ligandmolecules may be conjugated to oligonucleotides at the 5′-position bythe use of a ligand-nucleoside phosphoramidite wherein the ligand islinked to the 5′-hydroxy group directly or indirectly via a linker. Suchligand-nucleoside phosphoramidites are typically used at the end of anautomated synthesis procedure to provide a ligand-conjugatedoligonucleotide bearing the ligand at the 5′-terminus.

Examples of modified internucleoside linkages or backbones include, forexample, phosphorothioates, chiral phosphorothioates,phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters,methyl and other alkyl phosphonates including 3′-alkylene phosphonatesand chiral phosphonates, phosphinates, phosphoramidates including3′-amino phosphoramidate and aminoalkylphosphoramidates,thionophosphoramidates, thionoalkylphosphonates,thionoalkylphosphotriesters, and boranophosphates having normal 3′-5′linkages, 2′-5′ linked analogs of these, and those having invertedpolarity wherein the adjacent pairs of nucleoside units are linked 3′-5′to 5′-3′ or 2′-5′ to 5′-2′. Various salts, mixed salts and free-acidforms are also included.

Representative United States patents relating to the preparation of theabove phosphorus-atom-containing linkages include, but are not limitedto, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243;5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717;5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677;5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253;5,571,799; 5,587,361; 5,625,050; and 5,697,248, each of which is hereinincorporated by reference.

Examples of modified internucleoside linkages or backbones that do notinclude a phosphorus atom therein (i.e., oligonucleosides) havebackbones that are formed by short chain alkyl or cycloalkyl intersugarlinkages, mixed heteroatom and alkyl or cycloalkyl intersugar linkages,or one or more short chain heteroatomic or heterocyclic intersugarlinkages. These include those having morpholino linkages (formed in partfrom the sugar portion of a nucleoside); siloxane backbones; sulfide,sulfoxide and sulfone backbones; formacetyl and thioformacetylbackbones; methylene formacetyl and thioformacetyl backbones; alkenecontaining backbones; sulfamate backbones; methyleneimino andmethylenehydrazino backbones; sulfonate and sulfonamide backbones; amidebackbones; and others having mixed N, O, S and CH₂ component parts.

Representative United States patents relating to the preparation of theabove oligonucleosides include, but are not limited to, U.S. Pat. Nos.5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289;5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312;5,633,360; 5,677,437; and 5,677,439, each of which is hereinincorporated by reference.

In certain instances, the oligonucleotide may be modified by anon-ligand group. A number of non-ligand molecules have been conjugatedto oligonucleotides in order to enhance the activity, cellulardistribution or cellular uptake of the oligonucleotide, and proceduresfor performing such conjugations are available in the scientificliterature. Such non-ligand moieties have included lipid moieties, suchas cholesterol (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989,86:6553), cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994,4:1053), a thioether, e.g., hexyl-5-tritylthiol (Manoharan et al., Ann.N.Y. Acad. Sci., 1992, 660:306; Manoharan et al., Bioorg. Med. Chem.Let., 1993, 3:2765), a thiocholesterol (Oberhauser et al., Nucl. AcidsRes., 1992, 20:533), an aliphatic chain, e.g., dodecandiol or undecylresidues (Saison-Behmoaras et al., EMBO J., 1991, 10:111; Kabanov etal., FEBS Lett., 1990, 259:327; Svinarchuk et al., Biochimie, 1993,75:49), a phospholipid, e.g., di-hexadecyl-rac-glycerol ortriethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate(Manoharan et al., Tetrahedron Lett., 1995, 36:3651; Shea et al., Nucl.Acids Res., 1990, 18:3777), a polyamine or a polyethylene glycol chain(Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969), oradamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995,36:3651), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta,1995, 1264:229), or an octadecylamine orhexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol.Exp. Ther., 1996, 277:923). Representative United States patents thatteach the preparation of such oligonucleotide conjugates have beenlisted above. Typical conjugation protocols involve the synthesis ofoligonucleotides bearing an aminolinker at one or more positions of thesequence. The amino group is then reacted with the molecule beingconjugated using appropriate coupling or activating reagents. Theconjugation reaction may be performed either with the oligonucleotidestill bound to the solid support or following cleavage of theoligonucleotide in solution phase. Purification of the oligonucleotideconjugate by HPLC typically affords the pure conjugate. The use of acholesterol conjugate is particularly preferred since such a moiety canincrease targeting liver cells, a site of PCSK9 expression.

Vector Encoded RNAi Agents

In another aspect of the invention, PCSK9 specific dsRNA molecules thatmodulate PCSK9 gene expression activity are expressed from transcriptionunits inserted into DNA or RNA vectors (see, e.g., Couture, A, et al.,TIG. (1996), 12:5-10; Skillern, A., et al., International PCTPublication No. WO 00/22113, Conrad, International PCT Publication No.WO 00/22114, and Conrad, U.S. Pat. No. 6,054,299). These transgenes canbe introduced as a linear construct, a circular plasmid, or a viralvector, which can be incorporated and inherited as a transgeneintegrated into the host genome. The transgene can also be constructedto permit it to be inherited as an extrachromosomal plasmid (Gassmann,et al., Proc. Natl. Acad. Sci. USA (1995) 92:1292).

The individual strands of a dsRNA can be transcribed by promoters on twoseparate expression vectors and co-transfected into a target cell.Alternatively each individual strand of the dsRNA can be transcribed bypromoters both of which are located on the same expression plasmid. Inone embodiment, a dsRNA is expressed as an inverted repeat joined by alinker polynucleotide sequence such that the dsRNA has a stem and loopstructure.

The recombinant dsRNA expression vectors are generally DNA plasmids orviral vectors. dsRNA expressing viral vectors can be constructed basedon, but not limited to, adeno-associated virus (for a review, seeMuzyczka, et al., Curr. Topics Micro. Immunol. (1992) 158:97-129));adenovirus (see, for example, Berkner, et al., BioTechniques (1998)6:616), Rosenfeld et al. (1991, Science 252:431-434), and Rosenfeld etal. (1992), Cell 68:143-155)); or alphavirus as well as others known inthe art. Retroviruses have been used to introduce a variety of genesinto many different cell types, including epithelial cells, in vitroand/or in vivo (see, e.g., Eglitis, et al., Science (1985)230:1395-1398; Danos and Mulligan, Proc. Natl. Acad. Sci. USA (1998)85:6460-6464; Wilson et al., 1988, Proc. Natl. Acad. Sci. USA85:3014-3018; Armentano et al., 1990, Proc. Natl. Acad. Sci. USA87:61416145; Huber et al., 1991, Proc. Natl. Acad. Sci. USA88:8039-8043; Ferry et al., 1991, Proc. Natl. Acad. Sci. USA88:8377-8381; Chowdhury et al., 1991, Science 254:1802-1805; vanBeusechem. et al., 1992, Proc. Nad. Acad. Sci. USA 89:7640-19; Kay etal., 1992, Human Gene Therapy 3:641-647; Dai et al., 1992, Proc. Natl.Acad. Sci. USA 89:10892-10895; Hwu et al., 1993, J. Immunol.150:4104-4115; U.S. Pat. No. 4,868,116; U.S. Pat. No. 4,980,286; PCTApplication WO 89/07136; PCT Application WO 89/02468; PCT Application WO89/05345; and PCT Application WO 92/07573). Recombinant retroviralvectors capable of transducing and expressing genes inserted into thegenome of a cell can be produced by transfecting the recombinantretroviral genome into suitable packaging cell lines such as PA317 andPsi-CRIP (Comette et al., 1991, Human Gene Therapy 2:5-10; Cone et al.,1984, Proc. Natl. Acad. Sci. USA 81:6349). Recombinant adenoviralvectors can be used to infect a wide variety of cells and tissues insusceptible hosts (e.g., rat, hamster, dog, and chimpanzee) (Hsu et al.,1992, J. Infectious Disease, 166:769), and also have the advantage ofnot requiring mitotically active cells for infection.

Any viral vector capable of accepting the coding sequences for the dsRNAmolecule(s) to be expressed can be used, for example vectors derivedfrom adenovirus (AV); adeno-associated virus (AAV); retroviruses (e.g.,lentiviruses (LV), Rhabdoviruses, murine leukemia virus); herpes virus,and the like. The tropism of viral vectors can be modified bypseudotyping the vectors with envelope proteins or other surfaceantigens from other viruses, or by substituting different viral capsidproteins, as appropriate.

For example, lentiviral vectors of the invention can be pseudotyped withsurface proteins from vesicular stomatitis virus (VSV), rabies, Ebola,Mokola, and the like. AAV vectors of the invention can be made to targetdifferent cells by engineering the vectors to express different capsidprotein serotypes. For example, an AAV vector expressing a serotype 2capsid on a serotype 2 genome is called AAV 2/2. This serotype 2 capsidgene in the AAV 2/2 vector can be replaced by a serotype 5 capsid geneto produce an AAV 2/5 vector. Techniques for constructing AAV vectorswhich express different capsid protein serotypes are within the skill inthe art; see, e.g., Rabinowitz J E et al. (2002), J Virol 76:791-801,the entire disclosure of which is herein incorporated by reference.

Selection of recombinant viral vectors suitable for use in theinvention, methods for inserting nucleic acid sequences for expressingthe dsRNA into the vector, and methods of delivering the viral vector tothe cells of interest are within the skill in the art. See, for example,Dornburg R (1995), Gene Therap. 2: 301-310; Eglitis M A (1988),Biotechniques 6: 608-614; Miller A D (1990), Hum Gene Therap. 1: 5-14;Anderson W F (1998), Nature 392: 25-30; and Rubinson D A et al., Nat.Genet. 33: 401-406, the entire disclosures of which are hereinincorporated by reference.

Preferred viral vectors are those derived from AV and AAV. In aparticularly preferred embodiment, the dsRNA of the invention isexpressed as two separate, complementary single-stranded RNA moleculesfrom a recombinant AAV vector having, for example, either the U6 or H1RNA promoters, or the cytomegalovirus (CMV) promoter.

A suitable AV vector for expressing the dsRNA of the invention, a methodfor constructing the recombinant AV vector, and a method for deliveringthe vector into target cells, are described in Xia H et al. (2002), Nat.Biotech. 20: 1006-1010.

Suitable AAV vectors for expressing the dsRNA of the invention, methodsfor constructing the recombinant AV vector, and methods for deliveringthe vectors into target cells are described in Samulski R et al. (1987),J. Virol. 61: 3096-3101; Fisher K J et al. (1996), J. Virol, 70:520-532; Samulski R et al. (1989), J. Virol. 63: 3822-3826; U.S. Pat.No. 5,252,479; U.S. Pat. No. 5,139,941; International Patent ApplicationNo. WO 94/13788; and International Patent Application No. WO 93/24641,the entire disclosures of which are herein incorporated by reference.

The promoter driving dsRNA expression in either a DNA plasmid or viralvector of the invention may be a eukaryotic RNA polymerase I (e.g.ribosomal RNA promoter), RNA polymerase II (e.g. CMV early promoter oractin promoter or U1 snRNA promoter) or generally RNA polymerase IIIpromoter (e.g. U6 snRNA or 7SK RNA promoter) or a prokaryotic promoter,for example the T7 promoter, provided the expression plasmid alsoencodes T7 RNA polymerase required for transcription from a T7 promoter.The promoter can also direct transgene expression to the pancreas (see,e.g., the insulin regulatory sequence for pancreas (Bucchini et al.,1986, Proc. Natl. Acad. Sci. USA 83:2511-2515)).

In addition, expression of the transgene can be precisely regulated, forexample, by using an inducible regulatory sequence and expressionsystems such as a regulatory sequence that is sensitive to certainphysiological regulators, e.g., circulating glucose levels, or hormones(Docherty et al., 1994, FASEB J. 8:20-24). Such inducible expressionsystems, suitable for the control of transgene expression in cells or inmammals include regulation by ecdysone, by estrogen, progesterone,tetracycline, chemical inducers of dimerization, andisopropyl-beta-D1-thiogalactopyranoside (EPTG). A person skilled in theart would be able to choose the appropriate regulatory/promoter sequencebased on the intended use of the dsRNA transgene.

Generally, recombinant vectors capable of expressing dsRNA molecules aredelivered as described below, and persist in target cells.Alternatively, viral vectors can be used that provide for transientexpression of dsRNA molecules. Such vectors can be repeatedlyadministered as necessary. Once expressed, the dsRNAs bind to target RNAand modulate its function or expression. Delivery of dsRNA expressingvectors can be systemic, such as by intravenous or intramuscularadministration, by administration to target cells ex-planted from thepatient followed by reintroduction into the patient, or by any othermeans that allows for introduction into a desired target cell.

dsRNA expression DNA plasmids are typically transfected into targetcells as a complex with cationic lipid carriers (e.g. Oligofectamine) ornon-cationic lipid-based carriers (e.g. Transit-TKO™). Multiple lipidtransfections for dsRNA-mediated knockdowns targeting different regionsof a single PCSK9 gene or multiple PCSK9 genes over a period of a weekor more are also contemplated by the invention. Successful introductionof the vectors of the invention into host cells can be monitored usingvarious known methods. For example, transient transfection. can besignaled with a reporter, such as a fluorescent marker, such as GreenFluorescent Protein (GFP). Stable transfection of ex vivo cells can beensured using markers that provide the transfected cell with resistanceto specific environmental factors (e.g., antibiotics and drugs), such ashygromycin B resistance.

The PCSK9 specific dsRNA molecules can also be inserted into vectors andused as gene therapy vectors for human patients. Gene therapy vectorscan be delivered to a subject by, for example, intravenous injection,local administration (see U.S. Pat. No. 5,328,470) or by stereotacticinjection (see e.g., Chen et al. (1994) Proc. Natl. Acad. Sci. USA91:3054-3057). The pharmaceutical preparation of the gene therapy vectorcan include the gene therapy vector in an acceptable diluent, or caninclude a slow release matrix in which the gene delivery vehicle isimbedded. Alternatively, where the complete gene delivery vector can beproduced intact from recombinant cells, e.g., retroviral vectors, thepharmaceutical preparation can include one or more cells which producethe gene delivery system.

Pharmaceutical Compositions Containing dsRNA

In one embodiment, the invention provides pharmaceutical compositionscontaining a dsRNA, as described herein, and a pharmaceuticallyacceptable carrier and methods of administering the same. Thepharmaceutical composition containing the dsRNA is useful for treating adisease or disorder associated with the expression or activity of aPCSK9 gene, such as pathological processes mediated by PCSK9 expression,e.g., hyperlipidemia. Such pharmaceutical compositions are formulatedbased on the mode of delivery.

Dosage

The pharmaceutical compositions featured herein are administered indosages sufficient to inhibit expression of PCSK9 genes. In general, asuitable dose of dsRNA will be in the range of 0.01 to 200.0 milligramsper kilogram body weight of the recipient per day, generally in therange of 1 to 50 mg per kilogram body weight per day. For example, thedsRNA can be administered at 0.01 mg/kg, 0.05 mg/kg, 0.5 mg/kg, 1 mg/kg,1.5 mg/kg, 2.0 mg/kg, 3.0 mg/kg, 5.0 mg/kg, 10 mg/kg, 20 mg/kg, 30mg/kg, 40 mg/kg, or 50 mg/kg per single dose.

In another embodiment, the dosage is between 0.01 and 0.2 mg/kg. Forexample, the dsRNA can be administered at a dose of 0.01 mg/kg, 0.02mg/kg, 0.03 mg/kg, 0.04 mg/kg, 0.05 mg/kg, 0.06 mg/kg, 0.07 mg/kg 0.08mg/kg 0.09 mg/kg, 0.10 mg/kg, 0.11 mg/kg, 0.12 mg/kg, 0.13 mg/kg, 0.14mg/kg, 0.15 mg/kg, 0.16 mg/kg, 0.17 mg/kg, 0.18 mg/kg, 0.19 mg/kg, or0.20 mg/kg.

In one embodiment, the dosage is between 0.2 mg/kg and 1.5 mg/kg. Forexample, the dsRNA can be administered at a dose of 0.2 mg/kg, 0.3mg/kg, 0.4 mg/kg, 0.5 mg/kg, 0.6 mg/kg, 0.7 mg/kg, 0.8 mg/kg, 0.9 mg/kg,1 mg/kg, 1.1 mg/kg, 1.2 mg/kg, 1.3 mg/kg, 1.4 mg/kg, or 1.5 mg/kg.

The dsRNA can be administered at a dose of 0.03, 0.1, 0.3, or 1.3, or3.0 mg/kg.

The pharmaceutical composition can be administered once daily, or thedsRNA may be administered as two, three, or more sub-doses atappropriate intervals throughout the day. The effect of a single dose onPCSK9 levels is long lasting, such that subsequent doses areadministered at not more than 7 day intervals, or at not more than 1, 2,3, or 4 week intervals.

In one embodiment the lipid formulated PCSK9 targeted dsRNA isadministered at a first dose of about 3 mg/kg followed by administeringat least one subsequent dose once a week, wherein the subsequent dose islower than the first dose, e.g., the subsequent dose is about 1.0 mg/kgor about 0.3 mg/kg. The subsequent dose can be administered, e.g., oncea week for four weeks.

In some embodiments the dsRNA is administered using continuous infusionor delivery through a controlled release formulation. In that case, thedsRNA contained in each sub-dose must be correspondingly smaller inorder to achieve the total daily dosage. The dosage unit can also becompounded for delivery over several days, e.g., using a conventionalsustained release formulation which provides sustained release of thedsRNA over a several day period. Sustained release formulations are wellknown in the art and are particularly useful for delivery of agents at aparticular site, such as could be used with the agents of the presentinvention. In this embodiment, the dosage unit contains a correspondingmultiple of the daily dose.

The skilled artisan will appreciate that certain factors may influencethe dosage and timing required to effectively treat a subject, includingbut not limited to the severity of the disease or disorder, previoustreatments, the general health and/or age of the subject, and otherdiseases present. Moreover, treatment of a subject with atherapeutically effective amount of a composition can include a singletreatment or a series of treatments. Estimates of effective dosages andin vivo half-lives for the individual dsRNAs encompassed by theinvention can be made using conventional methodologies or on the basisof in vivo testing using an appropriate animal model, as describedelsewhere herein.

Advances in mouse genetics have generated a number of mouse models forthe study of various human diseases, such as pathological processesmediated by PCSK9 expression. Such models are used for in vivo testingof dsRNA, as well as for determining a therapeutically effective dose. Asuitable mouse model is, for example, a mouse containing a plasmidexpressing human PCSK9. Another suitable mouse model is a transgenicmouse carrying a transgene that expresses human PCSK9.

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD50 (the dose lethal to 50% of thepopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD50/ED50.Compounds that exhibit high therapeutic indices are preferred.

The data obtained from cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofcompositions featured in the invention lies generally within a range ofcirculating concentrations that include the ED50 with little or notoxicity. The dosage may vary within this range depending upon thedosage form employed and the route of administration utilized. For anycompound used in the methods featured in the invention, thetherapeutically effective dose can be estimated initially from cellculture assays. A dose may be formulated in animal models to achieve acirculating plasma concentration range of the compound or, whenappropriate, of the polypeptide product of a target sequence (e.g.,achieving a decreased concentration of the polypeptide) that includesthe IC50 (i.e., the concentration of the test compound which achieves ahalf-maximal inhibition of symptoms) as determined in cell culture. Suchinformation can be used to more accurately determine useful doses inhumans. Levels in plasma may be measured, for example, by highperformance liquid chromatography.

In addition to their administration, as discussed above, the dsRNAsfeatured in the invention can be administered in combination with otherknown agents effective in treatment of pathological processes mediatedby target gene expression. In any event, the administering physician canadjust the amount and timing of dsRNA administration on the basis ofresults observed using standard measures of efficacy known in the art ordescribed herein.

Administration

The pharmaceutical compositions of the present invention may beadministered in a number of ways depending upon whether local orsystemic treatment is desired and upon the area to be treated.Administration may be topical, pulmonary, e.g., by inhalation orinsufflation of powders or aerosols, including by nebulizer;intratracheal, intranasal, epidermal and transdermal, and subdermal,oral or parenteral, e.g., subcutaneous.

Typically, when treating a mammal with hyperlipidemia, the dsRNAmolecules are administered systemically via parental means. Parenteraladministration includes intravenous, intra-arterial, subcutaneous,intraperitoneal or intramuscular injection or infusion; or intracranial,e.g., intraparenchymal, intrathecal or intraventricular, administration.For example, dsRNAs, conjugated or unconjugate or formulated with orwithout liposomes, can be administered intravenously to a patient. Forsuch, a dsRNA molecule can be formulated into compositions such assterile and non-sterile aqueous solutions, non-aqueous solutions incommon solvents such as alcohols, or solutions in liquid or solid oilbases. Such solutions also can contain buffers, diluents, and othersuitable additives. For parenteral, intrathecal, or intraventricularadministration, a dsRNA molecule can be formulated into compositionssuch as sterile aqueous solutions, which also can contain buffers,diluents, and other suitable additives (e.g., penetration enhancers,carrier compounds, and other pharmaceutically acceptable carriers).Formulations are described in more detail herein.

The dsRNA can be delivered in a manner to target a particular tissue,such as the liver (e.g., the hepatocytes of the liver).

Formulations

The pharmaceutical formulations of the present invention, which mayconveniently be presented in unit dosage form, may be prepared accordingto conventional techniques well known in the pharmaceutical industry.Such techniques include the step of bringing into association the activeingredients with the pharmaceutical carrier(s) or excipient(s). Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association the active ingredients with liquid carriers orfinely divided solid carriers or both, and then, if necessary, shapingthe product.

The compositions of the present invention may be formulated into any ofmany possible dosage forms such as, but not limited to, tablets,capsules, gel capsules, liquid syrups, soft gels, suppositories, andenemas. The compositions of the present invention may also be formulatedas suspensions in aqueous, non-aqueous or mixed media. Aqueoussuspensions may further contain substances which increase the viscosityof the suspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension may also contain stabilizers.

Pharmaceutical compositions of the present invention include, but arenot limited to, solutions, emulsions, and liposome-containingformulations. These compositions may be generated from a variety ofcomponents that include, but are not limited to, preformed liquids,self-emulsifying solids and self-emulsifying semisolids. In one aspectare formulations that target the liver when treating hepatic disorderssuch as hyperlipidemia.

In addition, dsRNA that target the PCSK9 gene can be formulated intocompositions containing the dsRNA admixed, encapsulated, conjugated, orotherwise associated with other molecules, molecular structures, ormixtures of nucleic acids. For example, a composition containing one ormore dsRNA agents that target the PCSK9 gene can contain othertherapeutic agents, such as other cancer therapeutics or one or moredsRNA compounds that target non-PCSK9 genes.

Oral, Parenteral, Topical, and Biologic Formulations

Compositions and formulations for oral administration include powders orgranules, microparticulates, nanoparticulates, suspensions or solutionsin water or non-aqueous media, capsules, gel capsules, sachets, tabletsor minitablets. Thickeners, flavoring agents, diluents, emulsifiers,dispersing aids or binders may be desirable. In some embodiments, oralformulations are those in which dsRNAs featured in the invention areadministered in conjunction with one or more penetration enhancerssurfactants and chelators. Suitable surfactants include fatty acidsand/or esters or salts thereof, bile acids and/or salts thereof.Suitable bile acids/salts include chenodeoxycholic acid (CDCA) andursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid,deoxycholic acid, glucholic acid, glycholic acid, glycodeoxycholic acid,taurocholic acid, taurodeoxycholic acid, sodiumtauro-24,25-dihydro-fusidate and sodium glycodihydrofusidate. Suitablefatty acids include arachidonic acid, undecanoic acid, oleic acid,lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid,stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate,monoolein, dilaurin, glyceryl 1-monocaprate,1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or amonoglyceride, a diglyceride or a pharmaceutically acceptable saltthereof (e.g., sodium). In some embodiments, combinations of penetrationenhancers are used, for example, fatty acids/salts in combination withbile acids/salts. One exemplary combination is the sodium salt of lauricacid, capric acid and UDCA. Further penetration enhancers includepolyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether. dsRNAsfeatured in the invention may be delivered orally, in granular formincluding sprayed dried particles, or complexed to form micro ornanoparticles. dsRNA complexing agents include poly-amino acids;polyimines; polyacrylates; polyalkylacrylates, polyoxethanes,polyalkylcyanoacrylates; cationized gelatins, albumins, starches,acrylates, polyethyleneglycols (PEG) and starches;polyalkylcyanoacrylates; DEAE-derivatized polyimines, pollulans,celluloses and starches. Suitable complexing agents include chitosan,N-trimethylchitosan, poly-L-lysine, polyhistidine, polyornithine,polyspermines, protamine, polyvinylpyridine,polythiodiethylaminomethylethylene P(TDAE), polyaminostyrene (e.g.,p-amino), poly(methylcyanoacrylate), poly(ethylcyanoacrylate),poly(butylcyanoacrylate), poly(isobutylcyanoacrylate),poly(isohexylcynaoacrylate), DEAE-methacrylate, DEAE-hexylacrylate,DEAE-acrylamide, DEAE-albumin and DEAE-dextran, polymethylacrylate,polyhexylacrylate, poly(D,L-lactic acid), poly(DL-lactic-co-glycolicacid (PLGA), alginate, and polyethyleneglycol (PEG). Oral formulationsfor dsRNAs and their preparation are described in detail in U.S. Pat.No. 6,887,906, U.S. Patent Publication. No. 20030027780, and U.S. Pat.No. 6,747,014, each of which is incorporated herein by reference.

Compositions and formulations for parenteral, intraparenchymal (into thebrain), intrathecal, intraventricular or intrahepatic administration mayinclude sterile aqueous solutions which may also contain buffers,diluents and other suitable additives such as, but not limited to,penetration enhancers, carrier compounds and other pharmaceuticallyacceptable carriers or excipients.

Pharmaceutical compositions and formulations for topical administrationmay include transdermal patches, ointments, lotions, creams, gels,drops, suppositories, sprays, liquids and powders. Conventionalpharmaceutical carriers, aqueous, powder or oily bases, thickeners andthe like may be necessary or desirable. Suitable topical formulationsinclude those in which the dsRNAs featured in the invention are inadmixture with a topical delivery agent such as lipids, liposomes, fattyacids, fatty acid esters, steroids, chelating agents and surfactants.Suitable lipids and liposomes include neutral (e.g.,dioleoylphosphatidyl DOPE ethanolamine, dimyristoylphosphatidyl cholineDMPC, distearolyphosphatidyl choline) negative (e.g.,dimyristoylphosphatidyl glycerol DMPG) and cationic (e.g.,dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidylethanolamine DOTMA). dsRNAs featured in the invention may beencapsulated within liposomes or may form complexes thereto, inparticular to cationic liposomes. Alternatively, dsRNAs may be complexedto lipids, in particular to cationic lipids. Suitable fatty acids andesters include but are not limited to arachidonic acid, oleic acid,eicosanoic acid, lauric acid, caprylic acid, capric acid, myristic acid,palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate,tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate,1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or aC₁₋₁₀ alkyl ester (e.g., isopropylmyristate IPM), monoglyceride,diglyceride or pharmaceutically acceptable salt thereof. Topicalformulations are described in detail in U.S. Pat. No. 6,747,014, whichis incorporated herein by reference. In addition, dsRNA molecules can beadministered to a mammal as biologic or abiologic means as described in,for example, U.S. Pat. No. 6,271,359. Abiologic delivery can beaccomplished by a variety of methods including, without limitation, (1)loading liposomes with a dsRNA acid molecule provided herein and (2)complexing a dsRNA molecule with lipids or liposomes to form nucleicacid-lipid or nucleic acid-liposome complexes. The liposome can becomposed of cationic and neutral lipids commonly used to transfect cellsin vitro. Cationic lipids can complex (e.g., charge-associate) withnegatively charged nucleic acids to form liposomes. Examples of cationicliposomes include, without limitation, lipofectin, lipofectamine,lipofectace, and DOTAP. Procedures for forming liposomes are well knownin the art. Liposome compositions can be formed, for example, fromphosphatidylcholine, dimyristoyl phosphatidylcholine, dipalmitoylphosphatidylcholine, dimyristoyl phosphatidylglycerol, or dioleoylphosphatidylethanolamine. Numerous lipophilic agents are commerciallyavailable, including Lipofectin™ (Invitrogen/Life Technologies,Carlsbad, Calif.) and Effectene™ (Qiagen, Valencia, Calif.). Inaddition, systemic delivery methods can be optimized using commerciallyavailable cationic lipids such as DDAB or DOTAP, each of which can bemixed with a neutral lipid such as DOPE or cholesterol. In some cases,liposomes such as those described by Templeton et al. (NatureBiotechnology, 15: 647-652 (1997)) can be used. In other embodiments,polycations such as polyethyleneimine can be used to achieve delivery invivo and ex vivo (Boletta et al., J. Am Soc. Nephrol. 7: 1728 (1996)).Additional information regarding the use of liposomes to deliver nucleicacids can be found in U.S. Pat. No. 6,271,359, PCT Publication WO96/40964 and Morrissey, D. et al. 2005. Nat Biotechnol. 23(8):1002-7.

Biologic delivery can be accomplished by a variety of methods including,without limitation, the use of viral vectors. For example, viral vectors(e.g., adenovirus and herpes virus vectors) can be used to deliver dsRNAmolecules to liver cells. Standard molecular biology techniques can beused to introduce one or more of the dsRNAs provided herein into one ofthe many different viral vectors previously developed to deliver nucleicacid to cells. These resulting viral vectors can be used to deliver theone or more dsRNAs to cells by, for example, infection.

Liposomal Formulations

There are many organized surfactant structures besides microemulsionsthat have been studied and used for the formulation of drugs. Theseinclude monolayers, micelles, bilayers and vesicles. Vesicles, such asliposomes, have attracted great interest because of their specificityand the duration of action they offer from the standpoint of drugdelivery. As used in the present invention, the term “liposome” means avesicle composed of amphiphilic lipids arranged in a spherical bilayeror bilayers.

Liposomes are unilamellar or multilamellar vesicles which have amembrane formed from a lipophilic material and an aqueous interior. Theaqueous portion contains the composition to be delivered. Cationicliposomes possess the advantage of being able to fuse to the cell wall.Non-cationic liposomes, although not able to fuse as efficiently withthe cell wall, are taken up by macrophages in vivo.

In order to cross intact mammalian skin, lipid vesicles must passthrough a series of fine pores, each with a diameter less than 50 nm,under the influence of a suitable transdermal gradient. Therefore, it isdesirable to use a liposome which is highly deformable and able to passthrough such fine pores.

Further advantages of liposomes include: liposomes obtained from naturalphospholipids are biocompatible and biodegradable; liposomes canincorporate a wide range of water and lipid soluble drugs; and liposomescan protect encapsulated drugs in their internal compartments frommetabolism and degradation (Rosoff, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 245). Important considerations in thepreparation of liposome formulations are the lipid surface charge,vesicle size and the aqueous volume of the liposomes.

Liposomes are useful for the transfer and delivery of active ingredientsto the site of action. Because the liposomal membrane is structurallysimilar to biological membranes, when liposomes are applied to a tissue,the liposomes start to merge with the cellular membranes and as themerging of the liposome and cell progresses, the liposomal contents areemptied into the cell where the active agent may act.

Liposomal formulations have been the focus of extensive investigation asthe mode of delivery for many drugs. There is growing evidence that fortopical administration, liposomes present several advantages over otherformulations. Such advantages include reduced side-effects related tohigh systemic absorption of the administered drug, increasedaccumulation of the administered drug at the desired target, and theability to administer a wide variety of drugs, both hydrophilic andhydrophobic, into the skin.

Several reports have detailed the ability of liposomes to deliver agentsincluding high-molecular weight DNA into the skin. Compounds includinganalgesics, antibodies, hormones and high-molecular weight DNAs havebeen administered to the skin. The majority of applications resulted inthe targeting of the upper epidermis

Liposomes fall into two broad classes. Cationic liposomes are positivelycharged liposomes which interact with the negatively charged DNAmolecules to form a stable complex. The positively charged DNA/liposomecomplex binds to the negatively charged cell surface and is internalizedin an endosome. Due to the acidic pH within the endosome, the liposomesare ruptured, releasing their contents into the cell cytoplasm (Wang etal., Biochem. Biophys. Res. Commun., 1987, 147, 980-985).

Liposomes which are pH-sensitive or negatively-charged, entrap DNArather than complex with it. Since both the DNA and the lipid aresimilarly charged, repulsion rather than complex formation occurs.Nevertheless, some DNA is entrapped within the aqueous interior of theseliposomes. pH-sensitive liposomes have been used to deliver DNA encodingthe thymidine kinase gene to cell monolayers in culture. Expression ofthe exogenous gene was detected in the target cells (Zhou et al.,Journal of Controlled Release, 1992, 19, 269-274).

One major type of liposomal composition includes phospholipids otherthan naturally-derived phosphatidylcholine. Neutral liposomecompositions, for example, can be formed from dimyristoylphosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC).Anionic liposome compositions generally are formed from dimyristoylphosphatidylglycerol, while anionic fusogenic liposomes are formedprimarily from dioleoyl phosphatidylethanolamine (DOPE). Another type ofliposomal composition is formed from phosphatidylcholine (PC) such as,for example, soybean PC, and egg PC. Another type is formed frommixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.

Several studies have assessed the topical delivery of liposomal drugformulations to the skin. Application of liposomes containing interferonto guinea pig skin resulted in a reduction of skin herpes sores whiledelivery of interferon via other means (e.g., as a solution or as anemulsion) were ineffective (Weiner et al., Journal of Drug Targeting,1992, 2, 405-410). Further, an additional study tested the efficacy ofinterferon administered as part of a liposomal formulation to theadministration of interferon using an aqueous system, and concluded thatthe liposomal formulation was superior to aqueous administration (duPlessis et al., Antiviral Research, 1992, 18, 259-265).

Non-ionic liposomal systems have also been examined to determine theirutility in the delivery of drugs to the skin, in particular systemscomprising non-ionic surfactant and cholesterol. Non-ionic liposomalformulations comprising Novasome™ I (glyceryldilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome™ II(glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) wereused to deliver cyclosporin-A into the dermis of mouse skin. Resultsindicated that such non-ionic liposomal systems were effective infacilitating the deposition of cyclosporin-A into different layers ofthe skin (Hu et al., S.T.P. Pharma. Sci., 1994, 4, 6, 466).

Liposomes also include “sterically stabilized” liposomes, a term which,as used herein, refers to liposomes comprising one or more specializedlipids that, when incorporated into liposomes, result in enhancedcirculation lifetimes relative to liposomes lacking such specializedlipids. Examples of sterically stabilized liposomes are those in whichpart of the vesicle-forming lipid portion of the liposome (A) comprisesone or more glycolipids, such as monosialoganglioside G_(M1), or (B) isderivatized with one or more hydrophilic polymers, such as apolyethylene glycol (PEG) moiety. While not wishing to be bound by anyparticular theory, it is thought in the art that, at least forsterically stabilized liposomes containing gangliosides, sphingomyelin,or PEG-derivatized lipids, the enhanced circulation half-life of thesesterically stabilized liposomes derives from a reduced uptake into cellsof the reticuloendothelial system (RES) (Allen et al., FEBS Letters,1987, 223, 42; Wu et al., Cancer Research, 1993, 53, 3765).

Various liposomes comprising one or more glycolipids are known in theart. Papahadjopoulos et al. (Ann. N.Y. Acad. Sci., 1987, 507, 64)reported the ability of monosialoganglioside G_(M1), galactocerebrosidesulfate and phosphatidylinositol to improve blood half-lives ofliposomes. These findings were expounded upon by Gabizon et al. (Proc.Natl. Acad. Sci. U.S.A., 1988, 85, 6949). U.S. Pat. No. 4,837,028 and WO88/04924, both to Allen et al., disclose liposomes comprising (1)sphingomyelin and (2) the ganglioside G_(M1) or a galactocerebrosidesulfate ester. U.S. Pat. No. 5,543,152 (Webb et al.) discloses liposomescomprising sphingomyelin. Liposomes comprising1,2-sn-dimyristoylphosphat-idylcholine are disclosed in WO 97/13499 (Limet al.).

Many liposomes comprising lipids derivatized with one or morehydrophilic polymers, and methods of preparation thereof, are known inthe art. Sunamoto et al. (Bull. Chem. Soc. Jpn., 1980, 53, 2778)described liposomes comprising a nonionic detergent, 2C_(1215G), thatcontains a PEG moiety. Illum et al. (FEBS Lett., 1984, 167, 79) notedthat hydrophilic coating of polystyrene particles with polymeric glycolsresults in significantly enhanced blood half-lives. Syntheticphospholipids modified by the attachment of carboxylic groups ofpolyalkylene glycols (e.g., PEG) are described by Sears (U.S. Pat. Nos.4,426,330 and 4,534,899). Klibanov et al. (FEBS Lett., 1990, 268, 235)described experiments demonstrating that liposomes comprisingphosphatidylethanolamine (PE) derivatized with PEG or PEG stearate havesignificant increases in blood circulation half-lives. Blume et al.(Biochimica et Biophysica Acta, 1990, 1029, 91) extended suchobservations to other PEG-derivatized phospholipids, e.g., DSPE-PEG,formed from the combination of distearoylphosphatidylethanolamine (DSPE)and PEG. Liposomes having covalently bound PEG moieties on theirexternal surface are described in European Patent No. EP 0 445 131 B1and WO 90/04384 to Fisher. Liposome compositions containing 1-20 molepercent of PE derivatized with PEG, and methods of use thereof, aredescribed by Woodle et al. (U.S. Pat. Nos. 5,013,556 and 5,356,633) andMartin et al. (U.S. Pat. No. 5,213,804 and European Patent No. EP 0 496813 B1). Liposomes comprising a number of other lipid-polymer conjugatesare disclosed in WO 91/05545 and U.S. Pat. No. 5,225,212 (both to Martinet al.) and in WO 94/20073 (Zalipsky et al.) Liposomes comprisingPEG-modified ceramide lipids are described in WO 96/10391 (Choi et al).U.S. Pat. No. 5,540,935 (Miyazaki et al.) and U.S. Pat. No. 5,556,948(Tagawa et al.) describe PEG-containing liposomes that can be furtherderivatized with functional moieties on their surfaces.

A number of liposomes comprising nucleic acids are known in the art. WO96/40062 to Thierry et al. discloses methods for encapsulating highmolecular weight nucleic acids in liposomes. U.S. Pat. No. 5,264,221 toTagawa et al. discloses protein-bonded liposomes and asserts that thecontents of such liposomes may include a dsRNA. U.S. Pat. No. 5,665,710to Rahman et al. describes certain methods of encapsulatingoligodeoxynucleotides in liposomes. WO 97/04787 to Love et al. disclosesliposomes comprising dsRNAs targeted to the raf gene.

Transfersomes are yet another type of liposomes and are highlydeformable lipid aggregates which are attractive candidates for drugdelivery vehicles. Transfersomes may be described as lipid dropletswhich are so highly deformable that they are easily able to penetratethrough pores which are smaller than the droplet. Transfersomes areadaptable to the environment in which they are used, e.g., they areself-optimizing (adaptive to the shape of pores in the skin),self-repairing, frequently reach their targets without fragmenting, andoften self-loading. To make transfersomes, it is possible to add surfaceedge-activators, usually surfactants, to a standard liposomalcomposition. Transfersomes have been used to deliver serum albumin tothe skin. The transfersome-mediated delivery of serum albumin has beenshown to be as effective as subcutaneous injection of a solutioncontaining serum albumin.

Surfactants find wide application in formulations such as emulsions(including microemulsions) and liposomes. The most common way ofclassifying and ranking the properties of the many different types ofsurfactants, both natural and synthetic, is by the use of thehydrophile/lipophile balance (HLB). The nature of the hydrophilic group(also known as the “head”) provides the most useful means forcategorizing the different surfactants used in formulations (Rieger, inPharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988,p. 285).

If the surfactant molecule is not ionized, it is classified as anonionic surfactant. Nonionic surfactants find wide application inpharmaceutical and cosmetic products and are usable over a wide range ofpH values. In general their HLB values range from 2 to about 18depending on their structure. Nonionic surfactants include nonionicesters such as ethylene glycol esters, propylene glycol esters, glycerylesters, polyglyceryl esters, sorbitan esters, sucrose esters, andethoxylated esters. Nonionic alkanolamides and ethers such as fattyalcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylatedblock polymers are also included in this class. The polyoxyethylenesurfactants are the most popular members of the nonionic surfactantclass.

If the surfactant molecule carries a negative charge when it isdissolved or dispersed in water, the surfactant is classified asanionic. Anionic surfactants include carboxylates such as soaps, acyllactylates, acyl amides of amino acids, esters of sulfuric acid such asalkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkylbenzene sulfonates, acyl isethionates, acyl taurates andsulfosuccinates, and phosphates. The most important members of theanionic surfactant class are the alkyl sulfates and the soaps.

If the surfactant molecule carries a positive charge when it isdissolved or dispersed in water, the surfactant is classified ascationic. Cationic surfactants include quaternary ammonium salts andethoxylated amines. The quaternary ammonium salts are the most usedmembers of this class.

If the surfactant molecule has the ability to carry either a positive ornegative charge, the surfactant is classified as amphoteric. Amphotericsurfactants include acrylic acid derivatives, substituted alkylamides,N-alkylbetaines and phosphatides.

The use of surfactants in drug products, formulations and in emulsionshas been reviewed (Rieger, in Pharmaceutical Dosage Forms, MarcelDekker, Inc., New York, N.Y., 1988, p. 285).

Nucleic Acid Lipid Particles

In one embodiment, a dsRNA featured in the invention is fullyencapsulated in the lipid formulation, e.g., to form a nucleicacid-lipid particle, e.g., Nucleic acid-lipid particles typicallycontain a cationic lipid, a non-cationic lipid, a sterol, and a lipidthat prevents aggregation of the particle (e.g., a PEG-lipid conjugate).Nucleic acid-lipid particles are extremely useful for systemicapplications, as they exhibit extended circulation lifetimes followingintravenous (i.v.) injection and accumulate at distal sites (e.g., sitesphysically separated from the administration site). In addition, thenucleic acids when present in the nucleic acid-lipid particles of thepresent invention are resistant in aqueous solution to degradation witha nuclease. Nucleic acid-lipid particles and their method of preparationare disclosed in, e.g., U.S. Pat. Nos. 5,976,567; 5,981,501; 6,534,484;6,586,410; 6,815,432; and PCT Publication No. WO 96/40964.

Nucleic acid-lipid particles can further include one or more additionallipids and/or other components such as cholesterol. Other lipids may beincluded in the liposome compositions for a variety of purposes, such asto prevent lipid oxidation or to attach ligands onto the liposomesurface. Any of a number of lipids may be present, includingamphipathic, neutral, cationic, and anionic lipids. Such lipids can beused alone or in combination. Specific examples of additional lipidcomponents that may be present are described herein.

Additional components that may be present in a nucleic acid-lipidparticle include bilayer stabilizing components such as polyamideoligomers (see, e.g., U.S. Pat. No. 6,320,017), peptides, proteins,detergents, lipid-derivatives, such as PEG coupled tophosphatidylethanolamine and PEG conjugated to ceramides (see, U.S. Pat.No. 5,885,613).

A nucleic acid-lipid particle can include one or more of a second aminolipid or cationic lipid, a neutral lipid, a sterol, and a lipid selectedto reduce aggregation of lipid particles during formation, which mayresult from steric stabilization of particles which preventscharge-induced aggregation during formation.

Nucleic acid-lipid particles include, e.g., a SPLP, pSPLP, and SNALP.The term “SNALP” refers to a stable nucleic acid-lipid particle,including SPLP. The term “SPLP” refers to a nucleic acid-lipid particlecomprising plasmid DNA encapsulated within a lipid vesicle. SPLPsinclude “pSPLP,” which include an encapsulated condensing agent-nucleicacid complex as set forth in PCT Publication No. WO 00/03683.

The particles of the present invention typically have a mean diameter ofabout 50 nm to about 150 nm, more typically about 60 nm to about 130 nm,more typically about 70 nm to about 110 nm, most typically about 70 nmto about 90 nm, and are substantially nontoxic

In one embodiment, the lipid to drug ratio (mass/mass ratio) (e.g.,lipid to dsRNA ratio) will be in the range of from about 1:1 to about50:1, from about 1:1 to about 25:1, from about 3:1 to about 15:1, fromabout 4:1 to about 10:1, from about 5:1 to about 9:1, or about 6:1 toabout 9:1, or about 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, or 33:1.

Cationic Lipids

The nucleic acid-lipid particles of the invention typically include acationic lipid. The cationic lipid may be, for example,N,N-dioleyl-N,N-dimethylammonium chloride (DODAC),N,N-distearyl-N,N-dimethylammonium bromide (DDAB),N-(I-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP),N-(I-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA),N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA),1,2-DiLinoleyloxy-N,N-dimethylaminopropane (DLinDMA),1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA),1,2-Dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP),1,2-Dilinoleyoxy-3-(dimethylamino)acetoxypropane (DLin-DAC),1,2-Dilinoleyoxy-3-morpholinopropane (DLin-MA),1,2-Dilinoleoyl-3-dimethylaminopropane (DLinDAP),1,2-Dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA),1-Linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP),1,2-Dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.C1),1,2-Dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.C1),1,2-Dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), or3-(N,N-Dilinoleylamino)-1,2-propanediol (DLinAP),3-(N,N-Dioleylamino)-1,2-propanedio (DOAP),1,2-Dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA),1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA),2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA) oranalogs thereof,(3aR,5s,6aS)—N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine(ALNY-100), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl4-(dimethylamino)butanoate (MC3), or a mixture thereof.

Other cationic lipids, which carry a net positive charge at aboutphysiological pH, in addition to those specifically described above, mayalso be included in lipid particles of the invention. Such cationiclipids include, but are not limited to, N,N-dioleyl-N,N-dimethylammoniumchloride (“DODAC”); N-(2,3-dioleyloxy)propyl-N,N—N-triethylammoniumchloride (“DOTMA”); N,N-distearyl-N,N-dimethylammonium bromide (“DDAB”);N-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (“DOTAP”);1,2-Dioleyloxy-3-trimethylaminopropane chloride salt (“DOTAP.C1”);3β-(N—(N′,N′-dimethylaminoethane)-carbamoyl)cholesterol (“DC-Chol”),N-(1-(2,3-dioleyloxy)propyl)-N-2-(sperminecarboxamido)ethyl)-N,N-dimethylammoniumtrifluoracetate (“DOSPA”), dioctadecylamidoglycyl carboxyspermine(“DOGS”), 1,2-dileoyl-sn-3-phosphoethanolamine (“DOPE”),1,2-dioleoyl-3-dimethylammonium propane (“DODAP”),N,N-dimethyl-2,3-dioleyloxy)propylamine (“DODMA”), andN-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammoniumbromide (“DMRIE”). Additionally, a number of commercial preparations ofcationic lipids can be used, such as, e.g., LIPOFECTIN (including DOTMAand DOPE, available from GIBCO/BRL), and LIPOFECTAMINE (comprising DOSPAand DOPE, available from GIBCO/BRL). In particular embodiments, acationic lipid is an amino lipid.

As used herein, the term “amino lipid” is meant to include those lipidshaving one or two fatty acid or fatty alkyl chains and an amino headgroup (including an alkylamino or dialkylamino group) that may beprotonated to form a cationic lipid at physiological pH.

Other amino lipids would include those having alternative fatty acidgroups and other dialkylamino groups, including those in which the alkylsubstituents are different (e.g., N-ethyl-N-methylamino-,N-propyl-N-ethylamino- and the like). For those embodiments in which R¹¹and R¹² are both long chain alkyl or acyl groups, they can be the sameor different. In general, amino lipids having less saturated acyl chainsare more easily sized, particularly when the complexes must be sizedbelow about 0.3 microns, for purposes of filter sterilization. Aminolipids containing unsaturated fatty acids with carbon chain lengths inthe range of C₁₄ to C₂₂ are preferred. Other scaffolds can also be usedto separate the amino group and the fatty acid or fatty alkyl portion ofthe amino lipid. Suitable scaffolds are known to those of skill in theart.

In certain embodiments, amino or cationic lipids of the invention haveat least one protonatable or deprotonatable group, such that the lipidis positively charged at a pH at or below physiological pH (e.g. pH7.4), and neutral at a second pH, preferably at or above physiologicalpH. It will, of course, be understood that the addition or removal ofprotons as a function of pH is an equilibrium process, and that thereference to a charged or a neutral lipid refers to the nature of thepredominant species and does not require that all of the lipid bepresent in the charged or neutral form. Lipids that have more than oneprotonatable or deprotonatable group, or which are zwiterrionic, are notexcluded from use in the invention.

In certain embodiments, protonatable lipids according to the inventionhave a pKa of the protonatable group in the range of about 4 to about11. Most preferred is pKa of about 4 to about 7, because these lipidswill be cationic at a lower pH formulation stage, while particles willbe largely (though not completely) surface neutralized at physiologicalpH around pH 7.4. One of the benefits of this pKa is that at least somenucleic acid associated with the outside surface of the particle willlose its electrostatic interaction at physiological pH and be removed bysimple dialysis; thus greatly reducing the particle's susceptibility toclearance.

One example of a cationic lipid is1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA). Synthesis andpreparation of nucleic acid-lipid particles including DLinDMA isdescribed in International application number PCT/CA2009/00496, filedApr. 15, 2009.

In one embodiment, the cationic lipid XTC(2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane) is used to preparenucleic acid-lipid particles. Synthesis of XTC is described in U.S.provisional patent application No. 61/107,998 filed on Oct. 23, 2008,which is herein incorporated by reference.

In one aspect, the cationic lipids have the structure

and salts or isomers thereof, wherein R₁ and R₂ are each independentlyfor each occurrence optionally substituted C₁₀-C₃₀ alkyl, optionallysubstituted C₁₀-C₃₀ alkenyl, optionally substituted C₁₀-C₃₀ alkynyl,optionally substituted C₁₀-C₃₀ acyl, or -linker-ligand; R₃ is H,optionally substituted C₁-C₁₀ alkyl, optionally substituted C₂-C₁₀alkenyl, optionally substituted C₂-C₁₀ alkynyl, alkylhetrocycle,alkylphosphate, alkylphosphorothioate, alkylphosphorodithioate,alkylphosphonates, alkylamines, hydroxyalkyls, ω-aminoalkyls,ω-(substituted)aminoalkyls, ω-phosphoalkyls, ω-thiophosphoalkyls,optionally substituted polyethylene glycol (PEG, mw 100-40K), optionallysubstituted mPEG (mw 120-40K), heteroaryl, heterocycle, orlinker-ligand; and E is C(O)O or OC(O). Synthesis and use of this lipidfamily is described in WO 2010/054401 (PCTUS2009/063927 filed on Nov.10, 2009. The cationic lipid MC3 is one embodiment of this family ofcationic lipids.

In another embodiment, the cationic lipid MC3((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate),(e.g., DLin-M-C3-DMA) is used to prepare nucleic acid-lipid particles.Synthesis of MC3 and MC3 comprising formulations are described, e.g., inU.S. Provisional Ser. No. 61/244,834, filed Sep. 22, 2009, and U.S.Provisional Ser. No. 61/185,800, filed Jun. 10, 2009, and U.S. patentapplication Ser. No. 12/813/448 filed on Jun. 10, 2010, which are herebyincorporated by reference.

In another embodiment, the cationic lipid ALNY-100((3aR,5s,6aS)—N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine)is used to prepare nucleic acid-lipid particles. Synthesis of ALNY-100is described in International patent application number PCT/US09/63933filed on Nov. 10, 2009, which is herein incorporated by reference.

FIG. 28 illustrates the structures of ALNY-100 and MC3.

The cationic lipid may comprise from about 20 mol % to about 70 mol % orabout 45-65 mol % or about 10, 20, 30, 40, 50, 60, or 70 mol % of thetotal lipid present in the particle.

Non-Cationic Lipids

The nucleic acid-lipid particles of the invention can include anon-cationic lipid. The non-cationic lipid may be an anionic lipid or aneutral lipid. Examples include but not limited to,distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine(DOPC), dipalmitoylphosphatidylcholine (DPPC),dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol(DPPG), dioleoyl-phosphatidylethanolamine (DOPE),palmitoyloleoylphosphatidylcholine (POPC),palmitoyloleoylphosphatidylethanolamine (POPE),dioleoyl-phosphatidylethanolamine4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE),distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethyl PE,16-O-dimethyl PE, 18-1-trans PE,1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), cholesterol, or amixture thereof.

Anionic lipids suitable for use in lipid particles of the inventioninclude, but are not limited to, phosphatidylglycerol, cardiolipin,diacylphosphatidylserine, diacylphosphatidic acid, N-dodecanoylphosphatidylethanoloamine, N-succinyl phosphatidylethanolamine,N-glutaryl phosphatidylethanolamine, lysylphosphatidylglycerol, andother anionic modifying groups joined to neutral lipids.

Neutral lipids, when present in the lipid particle, can be any of anumber of lipid species which exist either in an uncharged or neutralzwitterionic form at physiological pH. Such lipids include, for examplediacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide,sphingomyelin, dihydrosphingomyelin, cephalin, and cerebrosides. Theselection of neutral lipids for use in the particles described herein isgenerally guided by consideration of, e.g., liposome size and stabilityof the liposomes in the bloodstream. Preferably, the neutral lipidcomponent is a lipid having two acyl groups, (i.e.,diacylphosphatidylcholine and diacylphosphatidylethanolamine). Lipidshaving a variety of acyl chain groups of varying chain length and degreeof saturation are available or may be isolated or synthesized bywell-known techniques. In one group of embodiments, lipids containingsaturated fatty acids with carbon chain lengths in the range of C₁₄ toC₂₂ are preferred. In another group of embodiments, lipids with mono- ordi-unsaturated fatty acids with carbon chain lengths in the range of C₁₄to C₂₂ are used. Additionally, lipids having mixtures of saturated andunsaturated fatty acid chains can be used. Preferably, the neutrallipids used in the invention are DOPE, DSPC, POPC, or any relatedphosphatidylcholine. The neutral lipids useful in the invention may alsobe composed of sphingomyelin, dihydrosphingomyeline, or phospholipidswith other head groups, such as serine and inositol.

In one embodiment the non-cationic lipid isdistearoylphosphatidylcholine (DSPC). In another embodiment thenon-cationic lipid is dipalmitoylphosphatidylcholine (DPPC).

The non-cationic lipid may be from about 5 mol % to about 90 mol %,about 5 mol % to about 10 mol %, about 10 mol %, or about 58 mol % ifcholesterol is included, of the total lipid present in the particle.

Conjugated Lipids

Conjugated lipids can be used in nucleic acid-lipid particle to preventaggregation, including polyethylene glycol (PEG)-modified lipids,monosialoganglioside Gm1, and polyamide oligomers (“PAO”) such as(described in U.S. Pat. No. 6,320,017). Other compounds with uncharged,hydrophilic, steric-barrier moieties, which prevent aggregation duringformulation, like PEG, Gm1 or ATTA, can also be coupled to lipids foruse as in the methods and compositions of the invention. ATTA-lipids aredescribed, e.g., in U.S. Pat. No. 6,320,017, and PEG-lipid conjugatesare described, e.g., in U.S. Pat. Nos. 5,820,873, 5,534,499 and5,885,613. Typically, the concentration of the lipid component selectedto reduce aggregation is about 1 to 15% (by mole percent of lipids).

Specific examples of PEG-modified lipids (or lipid-polyoxyethyleneconjugates) that are useful in the invention can have a variety of“anchoring” lipid portions to secure the PEG portion to the surface ofthe lipid vesicle. Examples of suitable PEG-modified lipids includePEG-modified phosphatidylethanolamine and phosphatidic acid,PEG-ceramide conjugates (e.g., PEG-CerC14 or PEG-CerC20) which aredescribed in co-pending U.S. Ser. No. 08/486,214, incorporated herein byreference, PEG-modified dialkylamines and PEG-modified1,2-diacyloxypropan-3-amines. Particularly preferred are PEG-modifieddiacylglycerols and dialkylglycerols.

In embodiments where a sterically-large moiety such as PEG or ATTA areconjugated to a lipid anchor, the selection of the lipid anchor dependson what type of association the conjugate is to have with the lipidparticle. It is well known that mePEG(mw2000)-diastearoylphosphatidylethanolamine (PEG-DSPE) will remainassociated with a liposome until the particle is cleared from thecirculation, possibly a matter of days. Other conjugates, such asPEG-CerC20 have similar staying capacity. PEG-CerC14, however, rapidlyexchanges out of the formulation upon exposure to serum, with a T_(1/2)less than 60 minutes in some assays. As illustrated in U.S. patentapplication Ser. No. 08/486,214, at least three characteristicsinfluence the rate of exchange: length of acyl chain, saturation of acylchain, and size of the steric-barrier head group. Compounds havingsuitable variations of these features may be useful for the invention.For some therapeutic applications, it may be preferable for thePEG-modified lipid to be rapidly lost from the nucleic acid-lipidparticle in vivo and hence the PEG-modified lipid will possessrelatively short lipid anchors. In other therapeutic applications, itmay be preferable for the nucleic acid-lipid particle to exhibit alonger plasma circulation lifetime and hence the PEG-modified lipid willpossess relatively longer lipid anchors. Exemplary lipid anchors includethose having lengths of from about C₁₄ to about C₂₂, preferably fromabout C₁₄ to about C₁₆. In some embodiments, a PEG moiety, for examplean mPEG-NH₂, has a size of about 1000, 2000, 5000, 10,000, 15,000 or20,000 Daltons.

It should be noted that aggregation preventing compounds do notnecessarily require lipid conjugation to function properly. Free PEG orfree ATTA in solution may be sufficient to prevent aggregation. If theparticles are stable after formulation, the PEG or ATTA can be dialyzedaway before administration to a subject.

The conjugated lipid that inhibits aggregation of particles may be, forexample, a polyethyleneglycol (PEG)-lipid including, without limitation,a PEG-diacylglycerol (DAG), a PEG-dialkyloxypropyl (DAA), aPEG-phospholipid, a PEG-ceramide (Cer), or a mixture thereof. ThePEG-DAA conjugate may be, for example, a PEG-dilauryloxypropyl (Ci₂), aPEG-dimyristyloxypropyl (Ci₄), a PEG-dipalmityloxypropyl (Ci₆), or aPEG-distearyloxypropyl (C]₈). Additional conjugated lipids includepolyethylene glycol-didimyristoyl glycerol (C14-PEG or PEG-C14, wherePEG has an average molecular weight of 2000 Da) (PEG-DMG);(R)-2,3-bis(octadecyloxy)propyl1-(methoxy poly(ethyleneglycol)2000)propylcarbamate) (PEG-DSG);PEG-carbamoyl-1,2-dimyristyloxypropylamine, in which PEG has an averagemolecular weight of 2000 Da (PEG-cDMA);N-Acetylgalactosamine-((R)-2,3-bis(octadecyloxy)propyl1-(methoxypoly(ethylene glycol)2000)propylcarbamate)) (GalNAc-PEG-DSG); andpolyethylene glycol-dipalmitoylglycerol (PEG-DPG).

In one embodiment the conjugated lipid is PEG-DMG. In another embodimentthe conjugated lipid is PEG-cDMA. In still another embodiment theconjugated lipid is PEG-DPG. Alternatively the conjugated lipid isGalNAc-PEG-DSG.

The conjugated lipid that prevents aggregation of particles may be from0 mol % to about 20 mol % or about 0.5 to about 5.0 mol % or about 2 mol% of the total lipid present in the particle.

The sterol component of the lipid mixture, when present, can be any ofthose sterols conventionally used in the field of liposome, lipidvesicle or lipid particle preparation. A preferred sterol ischolesterol.

In some embodiments, the nucleic acid-lipid particle further includes asterol, e.g., a cholesterol at, e.g., about 10 mol % to about 60 mol %or about 25 to about 40 mol % or about 48 mol % of the total lipidpresent in the particle.

Lipoproteins

In one embodiment, the formulations of the invention further comprise anapolipoprotein. As used herein, the term “apolipoprotein” or“lipoprotein” refers to apolipoproteins known to those of skill in theart and variants and fragments thereof and to apolipoprotein agonists,analogues or fragments thereof described below.

Suitable apolipoproteins include, but are not limited to, ApoA-I,ApoA-II, ApoA-IV, ApoA-V and ApoE, and active polymorphic forms,isoforms, variants and mutants as well as fragments or truncated formsthereof. In certain embodiments, the apolipoprotein is a thiolcontaining apolipoprotein. “Thiol containing apolipoprotein” refers toan apolipoprotein, variant, fragment or isoform that contains at leastone cysteine residue. The most common thiol containing apolipoproteinsare ApoA-I Milano (ApoA-I_(M)) and ApoA-I Paris (ApoA-I_(P)) whichcontain one cysteine residue (Jia et al., 2002, Biochem. Biophys. Res.Comm. 297: 206-13; Bielicki and Oda, 2002, Biochemistry 41: 2089-96).ApoA-II, ApoE2 and ApoE3 are also thiol containing apolipoproteins.Isolated ApoE and/or active fragments and polypeptide analogues thereof,including recombinantly produced forms thereof, are described in U.S.Pat. Nos. 5,672,685; 5,525,472; 5,473,039; 5,182,364; 5,177,189;5,168,045; 5,116,739; the disclosures of which are herein incorporatedby reference. ApoE3 is disclosed in Weisgraber, et al., “Human Eapoprotein heterogeneity: cysteine-arginine interchanges in the aminoacid sequence of the apo-E isoforms,” J. Biol. Chem. (1981) 256:9077-9083; and Ral1, et al., “Structural basis for receptor bindingheterogeneity of apolipoprotein E from type III hyperlipoproteinemicsubjects,” Proc. Nat. Acad. Sci. (1982) 79: 4696-4700. (See also GenBankaccession number K00396.)

In certain embodiments, the apolipoprotein can be in its mature form, inits preproapolipoprotein form or in its proapolipoprotein form. Homo-and heterodimers (where feasible) of pro- and mature ApoA-I (Duverger etal., 1996, Arterioscler. Thromb. Vasc. Biol. 16(12):1424-29), ApoA-IMilano (Klon et al., 2000, Biophys. J. 79:(3) 1679-87; Franceschini etal., 1985, J. Biol. Chem. 260: 1632-35), ApoA-I Paris (Daum et al.,1999, J. Mol. Med. 77:614-22), ApoA-II (Shelness et al., 1985, J. Biol.Chem. 260(14):8637-46; Shelness et al., 1984, J. Biol. Chem.259(15):9929-35), ApoA-IV (Duverger et al., 1991, Euro. J. Biochem.201(2):373-83), and ApoE (McLean et al., 1983, J. Biol. Chem.258(14):8993-9000) can also be utilized within the scope of theinvention.

In certain embodiments, the apolipoprotein can be a fragment, variant orisoform of the apolipoprotein. The term “fragment” refers to anyapolipoprotein having an amino acid sequence shorter than that of anative apolipoprotein and which fragment retains the activity of nativeapolipoprotein, including lipid binding properties. By “variant” ismeant substitutions or alterations in the amino acid sequences of theapolipoprotein, which substitutions or alterations, e.g., additions anddeletions of amino acid residues, do not abolish the activity of nativeapolipoprotein, including lipid binding properties. Thus, a variant cancomprise a protein or peptide having a substantially identical aminoacid sequence to a native apolipoprotein provided herein in which one ormore amino acid residues have been conservatively substituted withchemically similar amino acids. Examples of conservative substitutionsinclude the substitution of at least one hydrophobic residue such asisoleucine, valine, leucine or methionine for another. Likewise, thepresent invention contemplates, for example, the substitution of atleast one hydrophilic residue such as, for example, between arginine andlysine, between glutamine and asparagine, and between glycine and serine(see U.S. Pat. Nos. 6,004,925, 6,037,323 and 6,046,166). The term“isoform” refers to a protein having the same, greater or partialfunction and similar, identical or partial sequence, and may or may notbe the product of the same gene and usually tissue specific (seeWeisgraber 1990, J. Lipid Res. 31(8):1503-11; Hixson and Powers 1991, J.Lipid Res. 32(9):1529-35; Lackner et al., 1985, J. Biol. Chem.260(2):703-6; Hoeg et al., 1986, J. Biol. Chem. 261(9):3911-4; Gordon etal., 1984, J. Biol. Chem. 259(1):468-74; Powell et al., 1987, Cell50(6):831-40; Aviram et al., 1998, Arterioscler. Thromb. Vase. Biol.18(10):1617-24; Aviram et al., 1998, J. Clin. Invest. 101(8):1581-90;Billecke et al., 2000, Drug Metab. Dispos. 28(11):1335-42; Draganov etal., 2000, J. Biol. Chem. 275(43):33435-42; Steinmetz and Utermann 1985,J. Biol. Chem. 260(4):2258-64; Widler et al., 1980, J. Biol. Chem.255(21):10464-71; Dyer et al., 1995, J. Lipid Res. 36(1):80-8; Sacre etal., 2003, FEBS Lett. 540(1-3):181-7; Weers, et al., 2003, Biophys.Chem. 100(1-3):481-92; Gong et al., 2002, J. Biol. Chem.277(33):29919-26; Ohta et al., 1984, J. Biol. Chem. 259(23):14888-93 andU.S. Pat. No. 6,372,886).

In certain embodiments, the methods and compositions of the presentinvention include the use of a chimeric construction of anapolipoprotein. For example, a chimeric construction of anapolipoprotein can be comprised of an apolipoprotein domain with highlipid binding capacity associated with an apolipoprotein domaincontaining ischemia reperfusion protective properties. A chimericconstruction of an apolipoprotein can be a construction that includesseparate regions within an apolipoprotein (i.e., homologousconstruction) or a chimeric construction can be a construction thatincludes separate regions between different apolipoproteins (i.e.,heterologous constructions). Compositions comprising a chimericconstruction can also include segments that are apolipoprotein variantsor segments designed to have a specific character (e.g., lipid binding,receptor binding, enzymatic, enzyme activating, antioxidant orreduction-oxidation property) (see Weisgraber 1990, J. Lipid Res.31(8):1503-11; Hixson and Powers 1991, J. Lipid Res. 32(9):1529-35;Lackner et al., 1985, J. Biol. Chem. 260(2):703-6; Hoeg et al., 1986, J.Biol. Chem. 261(9):3911-4; Gordon et al., 1984, J. Biol. Chem.259(1):468-74; Powell et al., 1987, Cell 50(6):831-40; Aviram et al.,1998, Arterioscler. Thromb. Vasc. Biol. 18(10):1617-24; Aviram et al.,1998, J. Clin. Invest. 101(8):1581-90; Billecke et al., 2000, DrugMetab. Dispos. 28(11):1335-42; Draganov et al., 2000, J. Biol. Chem.275(43):33435-42; Steinmetz and Utermann 1985, J. Biol. Chem.260(4):2258-64; Widler et al., 1980, J. Biol. Chem. 255(21):10464-71;Dyer et al., 1995, J. Lipid Res. 36(1):80-8; Sorenson et al., 1999,Arterioscler. Thromb. Vasc. Biol. 19(9):2214-25; Palgunachari 1996,Arterioscler. Throb. Vasc. Biol. 16(2):328-38: Thurberg et al., J. Biol.Chem. 271(11):6062-70; Dyer 1991, J. Biol. Chem. 266(23):150009-15; Hill1998, J. Biol. Chem. 273(47):30979-84).

Apolipoproteins utilized in the invention also include recombinant,synthetic, semi-synthetic or purified apolipoproteins. Methods forobtaining apolipoproteins or equivalents thereof, utilized by theinvention are well-known in the art. For example, apolipoproteins can beseparated from plasma or natural products by, for example, densitygradient centrifugation or immunoaffinity chromatography, or producedsynthetically, semi-synthetically or using recombinant DNA techniquesknown to those of the art (see, e.g., Mulugeta et al., 1998, J.Chromatogr. 798(1-2): 83-90; Chung et al., 1980, J. Lipid Res.21(3):284-91; Cheung et al., 1987, J. Lipid Res. 28(8):913-29; Persson,et al., 1998, J. Chromatogr. 711:97-109; U.S. Pat. Nos. 5,059,528,5,834,596, 5,876,968 and 5,721,114; and PCT Publications WO 86/04920 andWO 87/02062).

Apolipoproteins utilized in the invention further include apolipoproteinagonists such as peptides and peptide analogues that mimic the activityof ApoA-I, ApoA-I Milano (ApoA-I_(M)), ApoA-I Paris (ApoA-I_(P)),ApoA-II, ApoA-IV, and ApoE. For example, the apolipoprotein can be anyof those described in U.S. Pat. Nos. 6,004,925, 6,037,323, 6,046,166,and 5,840,688, the contents of which are incorporated herein byreference in their entireties.

Apolipoprotein agonist peptides or peptide analogues can be synthesizedor manufactured using any technique for peptide synthesis known in theart including, e.g., the techniques described in U.S. Pat. Nos.6,004,925, 6,037,323 and 6,046,166. For example, the peptides may beprepared using the solid-phase synthetic technique initially describedby Merrifield (1963, J. Am. Chem. Soc. 85:2149-2154). Other peptidesynthesis techniques may be found in Bodanszky et al., PeptideSynthesis, John Wiley & Sons, 2d Ed., (1976) and other referencesreadily available to those skilled in the art. A summary of polypeptidesynthesis techniques can be found in Stuart and Young, Solid PhasePeptide. Synthesis, Pierce Chemical Company, Rockford, Ill., (1984).Peptides may also be synthesized by solution methods as described in TheProteins, Vol. II, 3d Ed., Neurath et al., Eds., p. 105-237, AcademicPress, New York, N.Y. (1976). Appropriate protective groups for use indifferent peptide syntheses are described in the above-mentioned textsas well as in McOmie, Protective Groups in Organic Chemistry, PlenumPress, New York, N.Y. (1973). The peptides of the present inventionmight also be prepared by chemical or enzymatic cleavage from largerportions of, for example, apolipoprotein A-I.

In certain embodiments, the apolipoprotein can be a mixture ofapolipoproteins. In one embodiment, the apolipoprotein can be ahomogeneous mixture, that is, a single type of apolipoprotein. Inanother embodiment, the apolipoprotein can be a heterogeneous mixture ofapolipoproteins, that is, a mixture of two or more differentapolipoproteins. Embodiments of heterogeneous mixtures ofapolipoproteins can comprise, for example, a mixture of anapolipoprotein from an animal source and an apolipoprotein from asemi-synthetic source. In certain embodiments, a heterogeneous mixturecan comprise, for example, a mixture of ApoA-I and ApoA-I Milano. Incertain embodiments, a heterogeneous mixture can comprise, for example,a mixture of ApoA-I Milano and ApoA-I Paris. Suitable mixtures for usein the methods and compositions of the invention will be apparent to oneof skill in the art.

If the apolipoprotein is obtained from natural sources, it can beobtained from a plant or animal source. If the apolipoprotein isobtained from an animal source, the apolipoprotein can be from anyspecies. In certain embodiments, the apolipoprotein can be obtained froman animal source. In certain embodiments, the apolipoprotein can beobtained from a human source. In preferred embodiments of the invention,the apolipoprotein is derived from the same species as the individual towhich the apolipoprotein is administered.

Other Components

In numerous embodiments, amphipathic lipids are included in lipidparticles of the invention. “Amphipathic lipids” refer to any suitablematerial, wherein the hydrophobic portion of the lipid material orientsinto a hydrophobic phase, while the hydrophilic portion orients towardthe aqueous phase. Such compounds include, but are not limited to,phospholipids, aminolipids, and sphingolipids. Representativephospholipids include sphingomyelin, phosphatidylcholine,phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol,phosphatidic acid, palmitoyloleoyl phosphatdylcholine,lysophosphatidylcholine, lysophosphatidylethanolamine,dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine,distearoylphosphatidylcholine, or dilinoleylphosphatidylcholine. Otherphosphorus-lacking compounds, such as sphingolipids, glycosphingolipidfamilies, diacylglycerols, and β-acyloxyacids, can also be used.Additionally, such amphipathic lipids can be readily mixed with otherlipids, such as triglycerides and sterols.

Also suitable for inclusion in the lipid particles of the invention areprogrammable fusion lipids. Such lipid particles have little tendency tofuse with cell membranes and deliver their payload until a given signalevent occurs. This allows the lipid particle to distribute more evenlyafter injection into an organism or disease site before it starts fusingwith cells. The signal event can be, for example, a change in pH,temperature, ionic environment, or time. In the latter case, a fusiondelaying or “cloaking” component, such as an ATTA-lipid conjugate or aPEG-lipid conjugate, can simply exchange out of the lipid particlemembrane over time. Exemplary lipid anchors include those having lengthsof from about C₁₄ to about C₂₂, preferably from about C₁₄ to about C₁₆.In some embodiments, a PEG moiety, for example an mPEG-NH₂, has a sizeof about 1000, 2000, 5000, 10,000, 15,000 or 20,000 Daltons.

A lipid particle conjugated to a nucleic acid agent can also include atargeting moiety, e.g., a targeting moiety that is specific to a celltype or tissue. Targeting of lipid particles using a variety oftargeting moieties, such as ligands, cell surface receptors,glycoproteins, vitamins (e.g., riboflavin) and monoclonal antibodies,has been previously described (see, e.g., U.S. Pat. Nos. 4,957,773 and4,603,044). The targeting moieties can include the entire protein orfragments thereof. Targeting mechanisms generally require that thetargeting agents be positioned on the surface of the lipid particle insuch a manner that the targeting moiety is available for interactionwith the target, for example, a cell surface receptor. A variety ofdifferent targeting agents and methods are known and available in theart, including those described, e.g., in Sapra, P. and Allen, T M, Prog.Lipid Res. 42(5):439-62 (2003); and Abra, R M et al., J. Liposome Res.12:1-3, (2002).

The use of lipid particles, i.e., liposomes, with a surface coating ofhydrophilic polymer chains, such as polyethylene glycol (PEG) chains,for targeting has been proposed (Allen, et al., Biochimica et BiophysicaActa 1237: 99-108 (1995); DeFrees, et al., Journal of the AmericanChemistry Society 118: 6101-6104 (1996); Blume, et al., Biochimica etBiophysica Acta 1149: 180-184 (1993); Klibanov, et al., Journal ofLiposome Research 2: 321-334 (1992); U.S. Pat. No. 5,013,556; Zalipsky,Bioconjugate Chemistry 4: 296-299 (1993); Zalipsky, FEBS Letters 353:71-74 (1994); Zalipsky, in Stealth Liposomes Chapter 9 (Lasic andMartin, Eds) CRC Press, Boca Raton Fla. (1995). In one approach, aligand, such as an antibody, for targeting the lipid particle is linkedto the polar head group of lipids forming the lipid particle. In anotherapproach, the targeting ligand is attached to the distal ends of the PEGchains forming the hydrophilic polymer coating (Klibanov, et al.,Journal of Liposome Research 2: 321-334 (1992); Kirpotin et al., FEBSLetters 388: 115-118 (1996)).

Standard methods for coupling the target agents can be used. Forexample, phosphatidylethanolamine, which can be activated for attachmentof target agents, or derivatized lipophilic compounds, such aslipid-derivatized bleomycin, can be used. Antibody-targeted liposomescan be constructed using, for instance, liposomes that incorporateprotein A (see, Renneisen, et al., J. Bio. Chem., 265:16337-16342 (1990)and Leonetti, et al., Proc. Natl. Acad. Sci. (USA), 87:2448-2451 (1990).Other examples of antibody conjugation are disclosed in U.S. Pat. No.6,027,726, the teachings of which are incorporated herein by reference.Examples of targeting moieties can also include other proteins, specificto cellular components, including antigens associated with neoplasms ortumors. Proteins used as targeting moieties can be attached to theliposomes via covalent bonds (see, Heath, Covalent Attachment ofProteins to Liposomes, 149 Methods in Enzymology 111-119 (AcademicPress, Inc. 1987)). Other targeting methods include the biotin-avidinsystem.

Production of Nucleic Acid-Lipid Particles

In one embodiment, the nucleic acid-lipid particle formulations of theinvention are produced via an extrusion method or an in-line mixingmethod.

The extrusion method (also referred to as preformed method or batchprocess) is a method where the empty liposomes (i.e. no nucleic acid)are prepared first, followed by the addition of nucleic acid to theempty liposome. Extrusion of liposome compositions through a small-porepolycarbonate membrane or an asymmetric ceramic membrane results in arelatively well-defined size distribution. Typically, the suspension iscycled through the membrane one or more times until the desired liposomecomplex size distribution is achieved. The liposomes may be extrudedthrough successively smaller-pore membranes, to achieve a gradualreduction in liposome size. In some instances, the lipid-nucleic acidcompositions which are formed can be used without any sizing. Thesemethods are disclosed in the U.S. Pat. No. 5,008,050; U.S. Pat. No.4,927,637; U.S. Pat. No. 4,737,323; Biochim Biophys Acta. 1979 Oct. 19;557(1):9-23; Biochim Biophys Acta. 1980 Oct. 2; 601(3):559-7; BiochimBiophys Acta. 1986 Jun. 13; 858(1):161-8; and Biochim. Biophys. Acta1985 812, 55-65, which are hereby incorporated by reference in theirentirety.

The in-line mixing method is a method wherein both the lipids and thenucleic acid are added in parallel into a mixing chamber. The mixingchamber can be a simple T-connector or any other mixing chamber that isknown to one skill in the art. These methods are disclosed in U.S. Pat.No. 6,534,018 and U.S. Pat. No. 6,855,277; US publication 2007/0042031and Pharmaceuticals Research, Vol. 22, No. 3, March 2005, p. 362-372,which are hereby incorporated by reference in their entirety.

It is further understood that the formulations of the invention can beprepared by any methods known to one of ordinary skill in the art.

Characterization of Nucleic Acid-Lipid Particles

Formulations prepared by either the standard or extrusion-free methodcan be characterized in similar manners. For example, formulations aretypically characterized by visual inspection. They should be whitishtranslucent solutions free from aggregates or sediment. Particle sizeand particle size distribution of lipid-nanoparticles can be measured bylight scattering using, for example, a Malvern Zetasizer Nano ZS(Malvern, USA). Particles should be about 20-300 nm, such as 40-100 nmin size. The particle size distribution should be unimodal. The totalsiRNA concentration in the formulation, as well as the entrappedfraction, is estimated using a dye exclusion assay. A sample of theformulated siRNA can be incubated with an RNA-binding dye, such asRibogreen (Molecular Probes) in the presence or absence of a formulationdisrupting surfactant, e.g., 0.5% Triton-X100. The total siRNA in theformulation can be determined by the signal from the sample containingthe surfactant, relative to a standard curve. The entrapped fraction isdetermined by subtracting the “free” siRNA content (as measured by thesignal in the absence of surfactant) from the total siRNA content.Percent entrapped siRNA is typically >85%. In one embodiment, theformulations of the invention are entrapped by at least 75%, at least80% or at least 90%.

For nucleic acid-lipid particle formulations, the particle size is atleast 30 nm, at least 40 nm, at least 50 nm, at least 60 nm, at least 70nm, at least 80 nm, at least 90 nm, at least 100 nm, at least 110 nm,and at least 120 nm. The suitable range is typically about at least 50nm to about at least 110 nm, about at least 60 nm to about at least 100nm, or about at least 80 nm to about at least 90 nm.

Formulations of Nucleic Acid-Lipid Particles

LNP01

One example of synthesis of a nucleic acid-lipid particle is as follows.Nucleic acid-lipid particles are synthesized using the lipidoidND98.4HCl (MW 1487) (Formula 1), Cholesterol (Sigma-Aldrich), andPEG-Ceramide C16 (Avanti Polar Lipids). This nucleic acid-lipid particleis sometimes referred to as a LNP01 particle. Stock solutions of each inethanol can be prepared as follows: ND98, 133 mg/ml; Cholesterol, 25mg/ml, PEG-Ceramide C16, 100 mg/ml. The ND98, Cholesterol, andPEG-Ceramide C16 stock solutions can then be combined in a, e.g.,42:48:10 molar ratio. The combined lipid solution can be mixed withaqueous siRNA (e.g., in sodium acetate pH 5) such that the final ethanolconcentration is about 35-45% and the final sodium acetate concentrationis about 100-300 mM. Lipid-siRNA nanoparticles typically formspontaneously upon mixing. Depending on the desired particle sizedistribution, the resultant nanoparticle mixture can be extruded througha polycarbonate membrane (e.g., 100 nm cut-off) using, for example, athermobarrel extruder, such as Lipex Extruder (Northern Lipids, Inc). Insome cases, the extrusion step can be omitted. Ethanol removal andsimultaneous buffer exchange can be accomplished by, for example,dialysis or tangential flow filtration. Buffer can be exchanged with,for example, phosphate buffered saline (PBS) at about pH 7, e.g., aboutpH 6.9, about pH 7.0, about pH 7.1, about pH 7.2, about pH 7.3, or aboutpH 7.4.

LNP01 formulations are described, e.g., in International ApplicationPublication No. WO 2008/042973, which is hereby incorporated byreference.

Additional exemplary nucleic acid-lipid particle formulations aredescribed in the following table. It is to be understood that the nameof the nucleic acid-lipid particle in the table is not meant to belimiting. For example, as used herein, the term SNALP refers toformulations that include the cationic lipid DLinDMA.

cationic lipid/non-cationic lipid/cholesterol/ PEG-lipid conjugate mol %ratio Name Lipid:siRNA ratio SNALP DLinDMA/DPPC/Cholesterol/PEG-cDMA(57.1/7.1/34.4/1.4) lipid:siRNA ~7:1 LNP-S-XXTC/DPPC/Cholesterol/PEG-cDMA 57.1/7.1/34.4/1.4 lipid:siRNA ~7:1 LNP05XTC/DSPC/Cholesterol/PEG-DMG 57.5/7.5/31.5/3.5 lipid:siRNA ~6:1 LNP06XTC/DSPC/Cholesterol/PEG-DMG 57.5/7.5/31.5/3.5 lipid:siRNA ~11:1 LNP07XTC/DSPC/Cholesterol/PEG-DMG 60/7.5/31/1.5, lipid:siRNA ~6:1 LNP08XTC/DSPC/Cholesterol/PEG-DMG 60/7.5/31/1.5, lipid:siRNA ~11:1 LNP09XTC/DSPC/Cholesterol/PEG-DMG 50/10/38.5/1.5 lipid:siRNA ~10:1 LNP10ALNY-100/DSPC/Cholesterol/PEG-DMG 50/10/38.5/1.5 lipid:siRNA ~10:1 LNP11MC3/DSPC/Cholesterol/PEG-DMG 50/10/38.5/1.5 lipid:siRNA ~10:1 LNP13XTC/DSPC/Cholesterol/PEG-DMG 50/10/38.5/1.5 lipid:siRNA ~33:1 LNP14MC3/DSPC/Cholesterol/PEG-DMG 40/15/40/5 lipid:siRNA ~11:1 LNP15MC3/DSPC/Cholesterol/PEG-DSG/GalNAc-PEG-DSG 50/10/35/4.5/0.5 lipid:siRNA~11:1 LNP16 MC3/DSPC/Cholesterol/PEG-DMG 50/10/38.5/1.5 lipid:siRNA ~7:1LNP17 MC3/DSPC/Cholesterol/PEG-DSG 50/10/38.5/1.5 lipid:siRNA ~10:1LNP18 MC3/DSPC/Cholesterol/PEG-DMG 50/10/38.5/1.5 lipid:siRNA ~12:1LNP19 MC3/DSPC/Cholesterol/PEG-DMG 50/10/35/5 lipid:siRNA ~8:1 LNP20MC3/DSPC/Cholesterol/PEG-DPG 50/10/38.5/1.5 lipid:siRNA ~10:1 LNP22XTC/DSPC/Cholesterol/PEG-DSG 50/10/38.5/1.5 lipid:siRNA ~10:1

XTC comprising formulations are described, e.g., in U.S. ProvisionalSer. No. 61/239,686, filed Sep. 3, 2009, which is hereby incorporated byreference.

MC3 comprising formulations are described, e.g., in U.S. ProvisionalSer. No. 61/244,834, filed Sep. 22, 2009, and U.S. Provisional Ser. No.61/185,800, filed Jun. 10, 2009, which are hereby incorporated byreference.

ALNY-100 comprising formulations are described, e.g., Internationalpatent application number PCT/US09/63933, filed on Nov. 10, 2009, whichis hereby incorporated by reference.

Additional representative formulations delineated in Tables 11 and 12.Lipid refers to a cationic lipid.

TABLE 11 Composition of exemplary nucleic acid-lipid particle (mole %)prepared via extrusion methods. Lipid (mol %) DSPC (mol %) Chol (mol %)PEG (mol %) Lipid/siRNA 20 30 40 10 2.13 20 30 40 10 2.35 20 30 40 102.37 20 30 40 10 3.23 20 30 40 10 3.91 30 20 40 10 2.89 30 20 40 10 3.3430 20 40 10 3.34 30 20 40 10 4.10 30 20 40 10 5.64 40 10 40 10 3.02 4010 40 10 3.35 40 10 40 10 3.74 40 10 40 10 5.80 40 10 40 10 8.00 45 5 4010 3.27 45 5 40 10 3.30 45 5 40 10 4.45 45 5 40 10 7.00 45 5 40 10 9.8050 0 40 10 27.03 20 35 40 5 3.00 20 35 40 5 3.32 20 35 40 5 3.05 20 3540 5 3.67 20 35 40 5 4.71 30 25 40 5 2.47 30 25 40 5 2.98 30 25 40 53.29 30 25 40 5 4.99 30 25 40 5 7.15 40 15 40 5 2.79 40 15 40 5 3.29 4015 40 5 4.33 40 15 40 5 7.05 40 15 40 5 9.63 45 10 40 5 2.44 45 10 40 53.21 45 10 40 5 4.29 45 10 40 5 6.50 45 10 40 5 8.67 20 35 40 5 4.10 2035 40 5 4.83 30 25 40 5 3.86 30 25 40 5 5.38 30 25 40 5 7.07 40 15 40 53.85 40 15 40 5 4.88 40 15 40 5 7.22 40 15 40 5 9.75 45 10 40 5 2.83 4510 40 5 3.85 45 10 40 5 4.88 45 10 40 5 7.05 45 10 40 5 9.29 45 20 30 54.01 45 20 30 5 3.70 50 15 30 5 4.75 50 15 30 5 3.80 55 10 30 5 3.85 5510 30 5 4.13 60 5 30 5 5.09 60 5 30 5 4.67 65 0 30 5 4.75 65 0 30 5 6.0656.5 10 30 3.5 3.70 56.5 10 30 3.5 3.56 57.5 10 30 2.5 3.48 57.5 10 302.5 3.20 58.5 10 30 1.5 3.24 58.5 10 30 1.5 3.13 59.5 10 30 0.5 3.2459.5 10 30 0.5 3.03 45 10 40 5 7.57 45 10 40 5 7.24 45 10 40 5 7.48 4510 40 5 7.84 65 0 30 5 4.01 60 5 30 5 3.70 55 10 30 5 3.65 50 10 35 53.43 50 15 30 5 3.80 45 15 35 5 3.70 45 20 30 5 3.75 45 25 25 5 3.85 5510 32.5 2.5 3.61 60 10 27.5 2.5 3.65 60 10 25 5 4.07 55 5 38.5 1.5 3.7560 10 28.5 1.5 3.43 55 10 33.5 1.5 3.48 60 5 33.5 1.5 3.43 55 5 37.5 2.53.75 60 5 32.5 2.5 4.52 60 5 32.5 2.5 3.52 45 15 (DMPC) 35 5 3.20 45 15(DPPC) 35 5 3.43 45 15 (DOPC) 35 5 4.52 45 15 (POPC) 35 5 3.85 55 5 37.52.5 3.96 55 10 32.5 2.5 3.56 60 5 32.5 2.5 3.80 60 10 27.5 2.5 3.75 60 530 5 4.19 60 5 33.5 1.5 3.48 60 5 33.5 1.5 6.64 60 5 30 5 3.90 60 5 30 54.65 60 5 30 5 5.88 60 5 30 5 7.51 60 5 30 5 9.51 60 5 30 5 11.06 62.52.5 50 5 6.63 45 15 35 5 3.31 45 15 35 5 6.80 60 5 25 10 6.48 60 5 32.52.5 3.43 60 5 30 5 3.90 60 5 30 5 7.61 45 15 35 5 3.13 45 15 35 5 6.4260 5 25 10 6.48 60 5 32.5 2.5 3.03 60 5 30 5 3.43 60 5 30 5 6.72 60 5 305 4.13 70 5 20 5 5.48 80 5 10 5 5.94 90 5 0 5 9.50 60 5 30 5 C12PEG 3.8560 5 30 5 3.70 60 5 30 5 C16PEG 3.80 60 5 30 5 4.19 60 5 29 5 4.07 60 530 5 3.56 60 5 30 5 3.39 60 5 30 5 3.96 60 5 30 5 4.01 60 5 30 5 4.07 605 30 5 4.25 60 5 30 5 3.80 60 5 30 5 3.31 60 5 30 5 4.83 60 5 30 5 4.6760 5 30 5 3.96 57.5 7.5 33.5 1.5 3.39 57.5 7.5 32.5 2.5 3.39 57.5 7.531.5 3.5 3.52 57.5 7.5 30 5 4.19 60 5 30 5 3.96 60 5 30 5 3.96 60 5 30 53.56 60 5 33.5 1.5 3.52 60 5 25 10 5.18 60 5 (DPPC) 30 5 4.25 60 5 32.52.5 3.70 57.5 7.5 31.5 3.5 3.06 57.5 7.5 31.5 3.5 3.65 57.5 7.5 31.5 3.54.70 57.5 7.5 31.5 3.5 6.56

TABLE 12 Composition of exemplary nucleic acid-lipid particles preparedvia in- line mixing DSPC Chol Lipid (mol %) (mol %) (mol %) PEG (mol %)Lipid A/siRNA 55 5 37.5 2.5 3.96 55 10 32.5 2.5 3.56 60 5 32.5 2.5 3.8060 10 27.5 2.5 3.75 60 5 30 5 4.19 60 5 33.5 1.5 3.48 60 5 33.5 1.5 6.6460 5 25 10 6.79 60 5 32.5 2.5 3.96 60 5 34 1 3.75 60 5 34.5 0.5 3.28 505 40 5 3.96 60 5 30 5 4.75 70 5 20 5 5.00 80 5 10 5 5.18 60 5 30 5 13.6060 5 30 5 14.51 60 5 30 5 6.20 60 5 30 5 4.60 60 5 30 5 6.20 60 5 30 55.82 40 5 54 1 3.39 40 7.5 51.5 1 3.39 40 10 49 1 3.39 50 5 44 1 3.39 507.5 41.5 1 3.43 50 10 39 1 3.35 60 5 34 1 3.52 60 7.5 31.5 1 3.56 60 1029 1 3.80 70 5 24 1 3.70 70 7.5 21.5 1 4.13 70 10 19 1 3.85 60 5 34 13.52 60 5 34 1 3.70 60 5 34 1 3.52 60 7.5 27.5 5 5.18 60 7.5 29 3.5 4.4560 5 31.5 3.5 4.83 60 7.5 31 1.5 3.48 57.5 7.5 30 5 4.75 57.5 7.5 31.53.5 4.83 57.5 5 34 3.5 4.67 57.5 7.5 33.5 1.5 3.43 55 7.5 32.5 5 4.38 557.5 34 3.5 4.13 55 5 36.5 3.5 4.38 55 7.5 36 1.5 3.35

Synthesis of Cationic Lipids.

Any of the compounds, e.g., cationic lipids and the like, used in thenucleic acid-lipid particles of the invention may be prepared by knownorganic synthesis techniques, including the methods described in moredetail in the Examples. All substituents are as defined below unlessindicated otherwise.

“Alkyl” means a straight chain or branched, noncyclic or cyclic,saturated aliphatic hydrocarbon containing from 1 to 24 carbon atoms.Representative saturated straight chain alkyls include methyl, ethyl,n-propyl, n-butyl, n-pentyl, n-hexyl, and the like; while saturatedbranched alkyls include isopropyl, sec-butyl, isobutyl, tert-butyl,isopentyl, and the like. Representative saturated cyclic alkyls includecyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like; whileunsaturated cyclic alkyls include cyclopentenyl and cyclohexenyl, andthe like.

“Alkenyl” means an alkyl, as defined above, containing at least onedouble bond between adjacent carbon atoms. Alkenyls include both cis andtrans isomers. Representative straight chain and branched alkenylsinclude ethylenyl, propylenyl, 1-butenyl, 2-butenyl, isobutylenyl,1-pentenyl, 2-pentenyl, 3-methyl-1-butenyl, 2-methyl-2-butenyl,2,3-dimethyl-2-butenyl, and the like.

“Alkynyl” means any alkyl or alkenyl, as defined above, whichadditionally contains at least one triple bond between adjacent carbons.Representative straight chain and branched alkynyls include acetylenyl,propynyl, 1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl, 3-methyl-1butynyl, and the like.

“Acyl” means any alkyl, alkenyl, or alkynyl wherein the carbon at thepoint of attachment is substituted with an oxo group, as defined below.For example, —C(═O)alkyl, —C(═O)alkenyl, and —C(═O)alkynyl are acylgroups.

“Heterocycle” means a 5- to 7-membered monocyclic, or 7- to 10-memberedbicyclic, heterocyclic ring which is either saturated, unsaturated, oraromatic, and which contains from 1 or 2 heteroatoms independentlyselected from nitrogen, oxygen and sulfur, and wherein the nitrogen andsulfur heteroatoms may be optionally oxidized, and the nitrogenheteroatom may be optionally quaternized, including bicyclic rings inwhich any of the above heterocycles are fused to a benzene ring. Theheterocycle may be attached via any heteroatom or carbon atom.Heterocycles include heteroaryls as defined below. Heterocycles includemorpholinyl, pyrrolidinonyl, pyrrolidinyl, piperidinyl, piperizynyl,hydantoinyl, valerolactamyl, oxiranyl, oxetanyl, tetrahydrofuranyl,tetrahydropyranyl, tetrahydropyridinyl, tetrahydroprimidinyl,tetrahydrothiophenyl, tetrahydrothiopyranyl, tetrahydropyrimidinyl,tetrahydrothiophenyl, tetrahydrothiopyranyl, and the like.

The terms “optionally substituted alkyl”, “optionally substitutedalkenyl”, “optionally substituted alkynyl”, “optionally substitutedacyl”, and “optionally substituted heterocycle” means that, whensubstituted, at least one hydrogen atom is replaced with a substituent.In the case of an oxo substituent (═O) two hydrogen atoms are replaced.In this regard, substituents include oxo, halogen, heterocycle, —CN,—OR^(x), —NR^(x)R^(y), —NR^(x)C(═O)R^(y), —NR^(x)SO₂R^(y), —C(═O)R^(x),—C(═O)OR^(x), —C(═O)NR^(x)R^(y), —SO_(n)R^(x) and —SO_(n)NR^(x)R^(y),wherein n is 0, 1 or 2, R^(x) and R^(y) are the same or different andindependently hydrogen, alkyl or heterocycle, and each of said alkyl andheterocycle substituents may be further substituted with one or more ofoxo, halogen, —OH, —CN, alkyl, —OR^(x), heterocycle, —NR^(x)R^(y),—NR^(x)C(═O)R^(y), —NR^(x)SO₂R^(y), —C(═O)R^(x), —C(═O)OR^(x),—C(═O)NR^(x)R^(y), —SO_(n)R^(x) and —SO_(n)NR^(x)R^(y).

“Halogen” means fluoro, chloro, bromo and iodo.

In some embodiments, the methods of the invention may require the use ofprotecting groups. Protecting group methodology is well known to thoseskilled in the art (see, for example, PROTECTIVE GROUPS IN ORGANICSYNTHESIS, Green, T. W. et al., Wiley-Interscience, New York City,1999). Briefly, protecting groups within the context of this inventionare any group that reduces or eliminates unwanted reactivity of afunctional group. A protecting group can be added to a functional groupto mask its reactivity during certain reactions and then removed toreveal the original functional group. In some embodiments an “alcoholprotecting group” is used. An “alcohol protecting group” is any groupwhich decreases or eliminates unwanted reactivity of an alcoholfunctional group. Protecting groups can be added and removed usingtechniques well known in the art.

Synthesis of MC3

Preparation of DLin-M-C3-DMA (i.e.,(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate)was as follows. A solution of(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-ol (0.53 g),4-N,N-dimethylaminobutyric acid hydrochloride (0.51 g),4-N,N-dimethylaminopyridine (0.61 g) and1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.53 g) indichloromethane (5 mL) was stirred at room temperature overnight. Thesolution was washed with dilute hydrochloric acid followed by diluteaqueous sodium bicarbonate. The organic fractions were dried overanhydrous magnesium sulphate, filtered and the solvent removed on arotovap. The residue was passed down a silica gel column (20 g) using a1-5% methanol/dichloromethane elution gradient. Fractions containing thepurified product were combined and the solvent removed, yielding acolorless oil (0.54 g). Further description is provided in WO2010/054401 (PCTUS2009/063927 filed on Nov. 10, 2009 and U.S. patentapplication Ser. No. 12/813/448 filed on Jun. 10, 2010.

Synthesis of Formula A

In one embodiment, nucleic acid-lipid particles of the invention areformulated using a cationic lipid of formula A:

where R1 and R2 are independently alkyl, alkenyl or alkynyl, each can beoptionally substituted, and R3 and R4 are independently lower alkyl orR3 and R4 can be taken together to form an optionally substitutedheterocyclic ring. In some embodiments, the cationic lipid is XTC(2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane). In general, thelipid of formula A above may be made by the following Reaction Schemes 1or 2, wherein all substituents are as defined above unless indicatedotherwise.

Lipid A, where R₁ and R₂ are independently alkyl, alkenyl or alkynyl,each can be optionally substituted, and R₃ and R₄ are independentlylower alkyl or R₃ and R₄ can be taken together to form an optionallysubstituted heterocyclic ring, can be prepared according to Scheme 1.Ketone 1 and bromide 2 can be purchased or prepared according to methodsknown to those of ordinary skill in the art. Reaction of 1 and 2 yieldsketal 3. Treatment of ketal 3 with amine 4 yields lipids of formula A.The lipids of formula A can be converted to the corresponding ammoniumsalt with an organic salt of formula 5, where X is anion counter ionselected from halogen, hydroxide, phosphate, sulfate, or the like.

Alternatively, the ketone 1 starting material can be prepared accordingto Scheme 2. Grignard reagent 6 and cyanide 7 can be purchased orprepared according to methods known to those of ordinary skill in theart. Reaction of 6 and 7 yields ketone 1. Conversion of ketone 1 to thecorresponding lipids of formula A is as described in Scheme 1.

Synthesis of ALNY-100

Synthesis of ketal 519 [ALNY-100] was performed using the followingscheme 3:

Synthesis of 515:

To a stirred suspension of LiAlH4 (3.74 g, 0.09852 mol) in 200 mlanhydrous THF in a two neck RBF (1 L), was added a solution of 514 (10g, 0.04926 mol) in 70 mL of THF slowly at 0° C. under nitrogenatmosphere. After complete addition, reaction mixture was warmed to roomtemperature and then heated to reflux for 4 h. Progress of the reactionwas monitored by TLC. After completion of reaction (by TLC) the mixturewas cooled to 0° C. and quenched with careful addition of saturatedNa2SO4 solution. Reaction mixture was stirred for 4 h at roomtemperature and filtered off. Residue was washed well with THF. Thefiltrate and washings were mixed and diluted with 400 mL dioxane and 26mL conc. HCl and stirred for 20 minutes at room temperature. Thevolatilities were stripped off under vacuum to furnish the hydrochloridesalt of 515 as a white solid. Yield: 7.12 g 1H-NMR (DMSO, 400 MHz):δ=9.34 (broad, 2H), 5.68 (s, 2H), 3.74 (m, 1H), 2.66-2.60 (m, 2H),2.50-2.45 (m, 5H).

Synthesis of 516:

To a stirred solution of compound 515 in 100 mL dry DCM in a 250 mL twoneck RBF, was added NEt3 (37.2 mL, 0.2669 mol) and cooled to 0° C. undernitrogen atmosphere. After a slow addition ofN-(benzyloxy-carbonyloxy)-succinimide (20 g, 0.08007 mol) in 50 mL dryDCM, reaction mixture was allowed to warm to room temperature. Aftercompletion of the reaction (2-3 h by TLC) mixture was washedsuccessively with 1N HCl solution (1×100 mL) and saturated NaHCO3solution (1×50 mL). The organic layer was then dried over anhyd. Na2SO4and the solvent was evaporated to give crude material which was purifiedby silica gel column chromatography to get 516 as sticky mass. Yield: 11g (89%). 1H-NMR (CDCl3, 400 MHz): δ=7.36-7.27 (m, 5H), 5.69 (s, 2H),5.12 (s, 2H), 4.96 (br., 1H) 2.74 (s, 3H), 2.60 (m, 2H), 2.30-2.25 (m,2H). LC-MS [M+H] −232.3 (96.94%).

Synthesis of 517A and 517B:

The cyclopentene 516 (5 g, 0.02164 mol) was dissolved in a solution of220 mL acetone and water (10:1) in a single neck 500 mL RBF and to itwas added N-methyl morpholine-N-oxide (7.6 g, 0.06492 mol) followed by4.2 mL of 7.6% solution of OsO4 (0.275 g, 0.00108 mol) in tert-butanolat room temperature. After completion of the reaction (˜3 h), themixture was quenched with addition of solid Na2SO3 and resulting mixturewas stirred for 1.5 h at room temperature. Reaction mixture was dilutedwith DCM (300 mL) and washed with water (2×100 mL) followed by saturatedNaHCO3 (1×50 mL) solution, water (1×30 mL) and finally with brine (1×50mL). Organic phase was dried over an.Na2SO4 and solvent was removed invacuum. Silica gel column chromatographic purification of the crudematerial was afforded a mixture of diastereomers, which were separatedby prep HPLC. Yield: −6 g crude

517A—Peak-1 (white solid), 5.13 g (96%). 1H-NMR (DMSO, 400 MHz):δ=7.39-7.31 (m, 5H), 5.04 (s, 2H), 4.78-4.73 (m, 1H), 4.48-4.47 (d, 2H),3.94-3.93 (m, 2H), 2.71 (s, 3H), 1.72-1.67 (m, 4H). LC-MS−[M+H]−266.3,[M+NH4+]−283.5 present, HPLC-97.86%. Stereochemistry confirmed by X-ray.

Synthesis of 518:

Using a procedure analogous to that described for the synthesis ofcompound 505, compound 518 (1.2 g, 41%) was obtained as a colorless oil.1H-NMR (CDCl3, 400 MHz): δ=7.35-7.33 (m, 4H), 7.30-7.27 (m, 1H),5.37-5.27 (m, 8H), 5.12 (s, 2H), 4.75 (m, 1H), 4.58-4.57 (m, 2H),2.78-2.74 (m, 7H), 2.06-2.00 (m, 8H), 1.96-1.91 (m, 2H), 1.62 (m, 4H),1.48 (m, 2H), 1.37-1.25 (br m, 36H), 0.87 (m, 6H). HPLC-98.65%.

General Procedure for the Synthesis of Compound 519:

A solution of compound 518 (1 eq) in hexane (15 mL) was added in adrop-wise fashion to an ice-cold solution of LAH in THF (1 M, 2 eq).After complete addition, the mixture was heated at 40 C over 0.5 h thencooled again on an ice bath. The mixture was carefully hydrolyzed withsaturated aqueous Na2SO4 then filtered through celite and reduced to anoil. Column chromatography provided the pure 519 (1.3 g, 68%) which wasobtained as a colorless oil. 13C NMR=130.2, 130.1 (×2), 127.9 (×3),112.3, 79.3, 64.4, 44.7, 38.3, 35.4, 31.5, 29.9 (×2), 29.7, 29.6 (×2),29.5 (×3), 29.3 (×2), 27.2 (×3), 25.6, 24.5, 23.3, 226, 14.1;Electrospray MS (+ve): Molecular weight for C44H80NO2 (M+H)+ Calc.654.6, Found 654.6.

Therapeutic Agent-Lipid Particle Compositions and Formulations

The invention includes compositions comprising a lipid particle of theinvention and an active agent, wherein the active agent is associatedwith the lipid particle. In particular embodiments, the active agent isa therapeutic agent. In particular embodiments, the active agent isencapsulated within an aqueous interior of the lipid particle. In otherembodiments, the active agent is present within one or more lipid layersof the lipid particle. In other embodiments, the active agent is boundto the exterior or interior lipid surface of a lipid particle.

“Fully encapsulated” as used herein indicates that the nucleic acid inthe particles is not significantly degraded after exposure to serum or anuclease assay that would significantly degrade free DNA. In a fullyencapsulated system, preferably less than 25% of particle nucleic acidis degraded in a treatment that would normally degrade 100% of freenucleic acid, more preferably less than 10% and most preferably lessthan 5% of the particle nucleic acid is degraded. Alternatively, fullencapsulation may be determined by an Oligreen® assay. Oligreen® is anultra-sensitive fluorescent nucleic acid stain for quantitatingoligonucleotides and single-stranded DNA in solution (available fromInvitrogen Corporation, Carlsbad, Calif.). Fully encapsulated alsosuggests that the particles are serum stable, that is, that they do notrapidly decompose into their component parts upon in vivoadministration.

Active agents, as used herein, include any molecule or compound capableof exerting a desired effect on a cell, tissue, organ, or subject. Sucheffects may be biological, physiological, or cosmetic, for example.Active agents may be any type of molecule or compound, including e.g.,nucleic acids, peptides and polypeptides, including, e.g., antibodies,such as, e.g., polyclonal antibodies, monoclonal antibodies, antibodyfragments; humanized antibodies, recombinant antibodies, recombinanthuman antibodies, and Primatized™ antibodies, cytokines, growth factors,apoptotic factors, differentiation-inducing factors, cell surfacereceptors and their ligands; hormones; and small molecules, includingsmall organic molecules or compounds.

In one embodiment, the active agent is a therapeutic agent, or a salt orderivative thereof. Therapeutic agent derivatives may be therapeuticallyactive themselves or they may be prodrugs, which become active uponfurther modification. Thus, in one embodiment, a therapeutic agentderivative retains some or all of the therapeutic activity as comparedto the unmodified agent, while in another embodiment, a therapeuticagent derivative lacks therapeutic activity.

In various embodiments, therapeutic agents include any therapeuticallyeffective agent or drug, such as anti-inflammatory compounds,anti-depressants, stimulants, analgesics, antibiotics, birth controlmedication, antipyretics, vasodilators, anti-angiogenics, cytovascularagents, signal transduction inhibitors, cardiovascular drugs, e.g.,anti-arrhythmic agents, vasoconstrictors, hormones, and steroids.

In certain embodiments, the therapeutic agent is an oncology drug, whichmay also be referred to as an anti-tumor drug, an anti-cancer drug, atumor drug, an antineoplastic agent, or the like. Examples of oncologydrugs that may be used according to the invention include, but are notlimited to, adriamycin, alkeran, allopurinol, altretamine, amifostine,anastrozole, araC, arsenic trioxide, azathioprine, bexarotene, biCNU,bleomycin, busulfan intravenous, busulfan oral, capecitabine (Xeloda),carboplatin, carmustine, CCNU, celecoxib, chlorambucil, cisplatin,cladribine, cyclosporin A, cytarabine, cytosine arabinoside,daunorubicin, cytoxan, daunorubicin, dexamethasone, dexrazoxane,dodetaxel, doxorubicin, doxorubicin, DTIC, epirubicin, estramustine,etoposide phosphate, etoposide and VP-16, exemestane, FK506,fludarabine, fluorouracil, 5-FU, gemcitabine (Gemzar),gemtuzumab-ozogamicin, goserelin acetate, hydrea, hydroxyurea,idarubicin, ifosfamide, imatinib mesylate, interferon, irinotecan(Camptostar, CPT-111), letrozole, leucovorin, leustatin, leuprolide,levamisole, litretinoin, megastrol, melphalan, L-PAM, mesna,methotrexate, methoxsalen, mithramycin, mitomycin, mitoxantrone,nitrogen mustard, paclitaxel, pamidronate, Pegademase, pentostatin,porfimer sodium, prednisone, rituxan, streptozocin, STI-571, tamoxifen,taxotere, temozolamide, teniposide, VM-26, topotecan (Hycamtin),toremifene, tretinoin, ATRA, valrubicin, velban, vinblastine,vincristine, VP16, and vinorelbine. Other examples of oncology drugsthat may be used according to the invention are ellipticin andellipticin analogs or derivatives, epothilones, intracellular kinaseinhibitors and camptothecins.

Additional Formulations

Emulsions

The compositions of the present invention may be prepared and formulatedas emulsions. Emulsions are typically heterogeneous systems of oneliquid dispersed in another in the form of droplets usually exceeding0.1 μm in diameter (Idson, in Pharmaceutical Dosage Forms, Lieberman,Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y.,volume 1, p. 199; Rosoff, in Pharmaceutical Dosage Forms, Lieberman,Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y.,Volume 1, p. 245; Block in Pharmaceutical Dosage Forms, Lieberman,Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y.,volume 2, p. 335; Higuchi et al., in Remington's PharmaceuticalSciences, Mack Publishing Co., Easton, Pa., 1985, p. 301). Emulsions areoften biphasic systems comprising two immiscible liquid phasesintimately mixed and dispersed with each other. In general, emulsionsmay be of either the water-in-oil (w/o) or the oil-in-water (o/w)variety. When an aqueous phase is finely divided into and dispersed asminute droplets into a bulk oily phase, the resulting composition iscalled a water-in-oil (w/o) emulsion. Alternatively, when an oily phaseis finely divided into and dispersed as minute droplets into a bulkaqueous phase, the resulting composition is called an oil-in-water (o/w)emulsion. Emulsions may contain additional components in addition to thedispersed phases, and the active drug which may be present as a solutionin either the aqueous phase, oily phase or itself as a separate phase.Pharmaceutical excipients such as emulsifiers, stabilizers, dyes, andanti-oxidants may also be present in emulsions as needed. Pharmaceuticalemulsions may also be multiple emulsions that are comprised of more thantwo phases such as, for example, in the case of oil-in-water-in-oil(o/w/o) and water-in-oil-in-water (w/o/w) emulsions. Such complexformulations often provide certain advantages that simple binaryemulsions do not. Multiple emulsions in which individual oil droplets ofan o/w emulsion enclose small water droplets constitute a w/o/wemulsion. Likewise a system of oil droplets enclosed in globules ofwater stabilized in an oily continuous phase provides an o/w/o emulsion.

Emulsions are characterized by little or no thermodynamic stability.Often, the dispersed or discontinuous phase of the emulsion is welldispersed into the external or continuous phase and maintained in thisform through the means of emulsifiers or the viscosity of theformulation. Either of the phases of the emulsion may be a semisolid ora solid, as is the case of emulsion-style ointment bases and creams.Other means of stabilizing emulsions entail the use of emulsifiers thatmay be incorporated into either phase of the emulsion. Emulsifiers maybroadly be classified into four categories: synthetic surfactants,naturally occurring emulsifiers, absorption bases, and finely dispersedsolids (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger andBanker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p.199).

Synthetic surfactants, also known as surface active agents, have foundwide applicability in the formulation of emulsions and have beenreviewed in the literature (Rieger, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 285; Idson, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), Marcel Dekker, Inc., New York,N.Y., 1988, volume 1, p. 199). Surfactants are typically amphiphilic andcomprise a hydrophilic and a hydrophobic portion. The ratio of thehydrophilic to the hydrophobic nature of the surfactant has been termedthe hydrophile/lipophile balance (HLB) and is a valuable tool incategorizing and selecting surfactants in the preparation offormulations. Surfactants may be classified into different classes basedon the nature of the hydrophilic group: nonionic, anionic, cationic andamphoteric (Rieger, in Pharmaceutical Dosage Forms, Lieberman, Riegerand Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1,p. 285).

Naturally occurring emulsifiers used in emulsion formulations includelanolin, beeswax, phosphatides, lecithin and acacia. Absorption basespossess hydrophilic properties such that they can soak up water to formw/o emulsions yet retain their semisolid consistencies, such asanhydrous lanolin and hydrophilic petrolatum. Finely divided solids havealso been used as good emulsifiers especially in combination withsurfactants and in viscous preparations. These include polar inorganicsolids, such as heavy metal hydroxides, non-swelling clays such asbentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidalaluminum silicate and colloidal magnesium aluminum silicate, pigmentsand nonpolar solids such as carbon or glyceryl tristearate.

Large varieties of non-emulsifying materials are also included inemulsion formulations and contribute to the properties of emulsions.These include fats, oils, waxes, fatty acids, fatty alcohols, fattyesters, humectants, hydrophilic colloids, preservatives and antioxidants(Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335;Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

Hydrophilic colloids or hydrocolloids include naturally occurring gumsand synthetic polymers such as polysaccharides (for example, acacia,agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth),cellulose derivatives (for example, carboxymethylcellulose andcarboxypropylcellulose), and synthetic polymers (for example, carbomers,cellulose ethers, and carboxyvinyl polymers). These disperse or swell inwater to form colloidal solutions that stabilize emulsions by formingstrong interfacial films around the dispersed-phase droplets and byincreasing the viscosity of the external phase.

Since emulsions often contain a number of ingredients such ascarbohydrates, proteins, sterols and phosphatides that may readilysupport the growth of microbes, these formulations often incorporatepreservatives. Commonly used preservatives included in emulsionformulations include methyl paraben, propyl paraben, quaternary ammoniumsalts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boricacid. Antioxidants are also commonly added to emulsion formulations toprevent deterioration of the formulation. Antioxidants used may be freeradical scavengers such as tocopherols, alkyl gallates, butylatedhydroxyanisole, butylated hydroxytoluene, or reducing agents such asascorbic acid and sodium metabisulfite, and antioxidant synergists suchas citric acid, tartaric acid, and lecithin.

The application of emulsion formulations via dermatological, oral andparenteral routes and methods for their manufacture has been reviewed inthe literature (Idson, in Pharmaceutical Dosage Forms, Lieberman, Riegerand Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1,p. 199). Emulsion formulations for oral delivery have been very widelyused because of ease of formulation, as well as efficacy from anabsorption and bioavailability standpoint (Rosoff, in PharmaceuticalDosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker,Inc., New York, N.Y., volume 1, p. 245; Idson, in Pharmaceutical DosageForms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc.,New York, N.Y., volume 1, p. 199). Mineral-oil base laxatives,oil-soluble vitamins and high fat nutritive preparations are among thematerials that have commonly been administered orally as o/w emulsions.

In one embodiment of the present invention, the compositions of dsRNAsand nucleic acids are formulated as microemulsions. A microemulsion maybe defined as a system of water, oil and amphiphile which is a singleoptically isotropic and thermodynamically stable liquid solution(Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245).Typically microemulsions are systems that are prepared by firstdispersing an oil in an aqueous surfactant solution and then adding asufficient amount of a fourth component, generally an intermediatechain-length alcohol to form a transparent system. Therefore,microemulsions have also been described as thermodynamically stable,isotropically clear dispersions of two immiscible liquids that arestabilized by interfacial films of surface-active molecules (Leung andShah, in: Controlled Release of Drugs: Polymers and Aggregate Systems,Rosoff, M., Ed., 1989, VCH Publishers, New York, pages 185-215).Microemulsions commonly are prepared via a combination of three to fivecomponents that include oil, water, surfactant, cosurfactant andelectrolyte. Whether the microemulsion is of the water-in-oil (w/o) oran oil-in-water (o/w) type is dependent on the properties of the oil andsurfactant used and on the structure and geometric packing of the polarheads and hydrocarbon tails of the surfactant molecules (Schott, inRemington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa.,1985, p. 271).

The phenomenological approach utilizing phase diagrams has beenextensively studied and has yielded a comprehensive knowledge, to oneskilled in the art, of how to formulate microemulsions (Rosoff, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Block, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335). Compared toconventional emulsions, microemulsions offer the advantage ofsolubilizing water-insoluble drugs in a formulation of thermodynamicallystable droplets that are formed spontaneously.

Surfactants used in the preparation of microemulsions include, but arenot limited to, ionic surfactants, non-ionic surfactants, Brij 96,polyoxyethylene oleyl ethers, polyglycerol fatty acid esters,tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310),hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500),decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750),decaglycerol sequioleate (SO750), decaglycerol decaoleate (DAO750),alone or in combination with cosurfactants. The cosurfactant, usually ashort-chain alcohol such as ethanol, 1-propanol, and 1-butanol, servesto increase the interfacial fluidity by penetrating into the surfactantfilm and consequently creating a disordered film because of the voidspace generated among surfactant molecules. Microemulsions may, however,be prepared without the use of cosurfactants and alcohol-freeself-emulsifying microemulsion systems are known in the art. The aqueousphase may typically be, but is not limited to, water, an aqueoussolution of the drug, glycerol, PEG300, PEG400, polyglycerols, propyleneglycols, and derivatives of ethylene glycol. The oil phase may include,but is not limited to, materials such as Captex 300, Captex 355, CapmulMCM, fatty acid esters, medium chain (C8-C12) mono, di, andtri-glycerides, polyoxyethylated glyceryl fatty acid esters, fattyalcohols, polyglycolized glycerides, saturated polyglycolized C8-C10glycerides, vegetable oils and silicone oil.

Microemulsions are particularly of interest from the standpoint of drugsolubilization and the enhanced absorption of drugs. Lipid basedmicroemulsions (both o/w and w/o) have been proposed to enhance the oralbioavailability of drugs, including peptides (Constantinides et al.,Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, Meth. Find. Exp.Clin. Pharmacol., 1993, 13, 205). Microemulsions afford advantages ofimproved drug solubilization, protection of drug from enzymatichydrolysis, possible enhancement of drug absorption due tosurfactant-induced alterations in membrane fluidity and permeability,ease of preparation, ease of oral administration over solid dosageforms, improved clinical potency, and decreased toxicity (Constantinideset al., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm.Sci., 1996, 85, 138-143). Often microemulsions may form spontaneouslywhen their components are brought together at ambient temperature. Thismay be particularly advantageous when formulating thermolabile drugs,peptides or dsRNAs. Microemulsions have also been effective in thetransdermal delivery of active components in both cosmetic andpharmaceutical applications. It is expected that the microemulsioncompositions and formulations of the present invention will facilitatethe increased systemic absorption of dsRNAs and nucleic acids from thegastrointestinal tract, as well as improve the local cellular uptake ofdsRNAs and nucleic acids.

Microemulsions of the present invention may also contain additionalcomponents and additives such as sorbitan monostearate (Grill 3),Labrasol, and penetration enhancers to improve the properties of theformulation and to enhance the absorption of the dsRNAs and nucleicacids of the present invention. Penetration enhancers used in themicroemulsions of the present invention may be classified as belongingto one of five broad categories—surfactants, fatty acids, bile salts,chelating agents, and non-chelating non-surfactants (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Eachof these classes has been discussed above.

Penetration Enhancers

In one embodiment, the present invention employs various penetrationenhancers to effect the efficient delivery of nucleic acids,particularly dsRNAs, to the skin of animals. Most drugs are present insolution in both ionized and nonionized forms. However, usually onlylipid soluble or lipophilic drugs readily cross cell membranes. It hasbeen discovered that even non-lipophilic drugs may cross cell membranesif the membrane to be crossed is treated with a penetration enhancer. Inaddition to aiding the diffusion of non-lipophilic drugs across cellmembranes, penetration enhancers also enhance the permeability oflipophilic drugs.

Penetration enhancers may be classified as belonging to one of fivebroad categories, i.e., surfactants, fatty acids, bile salts, chelatingagents, and non-chelating non-surfactants (Lee et al., Critical Reviewsin Therapeutic Drug Carrier Systems, 1991, p. 92). Each of the abovementioned classes of penetration enhancers are described below ingreater detail.

Surfactants: In connection with the present invention, surfactants (or“surface-active agents”) are chemical entities which, when dissolved inan aqueous solution, reduce the surface tension of the solution or theinterfacial tension between the aqueous solution and another liquid,with the result that absorption of dsRNAs through the mucosa isenhanced. In addition to bile salts and fatty acids, these penetrationenhancers include, for example, sodium lauryl sulfate,polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether) (Leeet al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92); and perfluorochemical emulsions, such as FC-43. Takahashi et al.,J. Pharm. Pharmacol., 1988, 40, 252).

Fatty acids: Various fatty acids and their derivatives which act aspenetration enhancers include, for example, oleic acid, lauric acid,capric acid (n-decanoic acid), myristic acid, palmitic acid, stearicacid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein(1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid,glycerol 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines,acylcholines, C₁₋₁₀ alkyl esters thereof (e.g., methyl, isopropyl andt-butyl), and mono- and di-glycerides thereof (i.e., oleate, laurate,caprate, myristate, palmitate, stearate, linoleate, etc.) (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92;Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990,7, 1-33; El Hariri et al., J. Pharm. Pharmacol., 1992, 44, 651-654).

Bile salts: The physiological role of bile includes the facilitation ofdispersion and absorption of lipids and fat-soluble vitamins (Brunton,Chapter 38 in: Goodman & Gilman's The Pharmacological Basis ofTherapeutics, 9th Ed., Hardman et al. Eds., McGraw-Hill, New York, 1996,pp. 934-935). Various natural bile salts, and their syntheticderivatives, act as penetration enhancers. Thus the term “bile salts”includes any of the naturally occurring components of bile as well asany of their synthetic derivatives. Suitable bile salts include, forexample, cholic acid (or its pharmaceutically acceptable sodium salt,sodium cholate), dehydrocholic acid (sodium dehydrocholate), deoxycholicacid (sodium deoxycholate), glucholic acid (sodium glucholate),glycholic acid (sodium glycocholate), glycodeoxycholic acid (sodiumglycodeoxycholate), taurocholic acid (sodium taurocholate),taurodeoxycholic acid (sodium taurodeoxycholate), chenodeoxycholic acid(sodium chenodeoxycholate), ursodeoxycholic acid (UDCA), sodiumtauro-24,25-dihydro-fusidate (STDHF), sodium glycodihydrofusidate andpolyoxyethylene-9-lauryl ether (POE) (Lee et al., Critical Reviews inTherapeutic Drug Carrier Systems, 1991, page 92; Swinyard, Chapter 39In: Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., MackPublishing Co., Easton, Pa., 1990, pages 782-783; Muranishi, CriticalReviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Yamamoto etal., J. Pharm. Exp. Ther., 1992, 263, 25; Yamashita et al., J. Pharm.Sci., 1990, 79, 579-583).

Chelating Agents: Chelating agents, as used in connection with thepresent invention, can be defined as compounds that remove metallic ionsfrom solution by forming complexes therewith, with the result thatabsorption of dsRNAs through the mucosa is enhanced. With regards totheir use as penetration enhancers in the present invention, chelatingagents have the added advantage of also serving as DNase inhibitors, asmost characterized DNA nucleases require a divalent metal ion forcatalysis and are thus inhibited by chelating agents (Jarrett, J.Chromatogr., 1993, 618, 315-339). Suitable chelating agents include butare not limited to disodium ethylenediaminetetraacetate (EDTA), citricacid, salicylates (e.g., sodium salicylate, 5-methoxysalicylate andhomovanilate), N-acyl derivatives of collagen, laureth-9 and N-aminoacyl derivatives of beta-diketones (enamines) (Lee et al., CriticalReviews in Therapeutic Drug Carrier Systems, 1991, page 92; Muranishi,Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33;Buur et al., J. Control Rel., 1990, 14, 43-51).

Non-chelating non-surfactants: As used herein, non-chelatingnon-surfactant penetration enhancing compounds can be defined ascompounds that demonstrate insignificant activity as chelating agents oras surfactants but that nonetheless enhance absorption of dsRNAs throughthe alimentary mucosa (Muranishi, Critical Reviews in Therapeutic DrugCarrier Systems, 1990, 7, 1-33). This class of penetration enhancersincludes, for example, unsaturated cyclic ureas, 1-alkyl- and1-alkenylazacyclo-alkanone derivatives (Lee et al., Critical Reviews inTherapeutic Drug Carrier Systems, 1991, page 92); and non-steroidalanti-inflammatory agents such as diclofenac sodium, indomethacin andphenylbutazone (Yamashita et al., J. Pharm. Pharmacol., 1987, 39,621-626).

Agents that enhance uptake of dsRNAs at the cellular level may also beadded to the pharmaceutical and other compositions of the presentinvention. For example, cationic lipids, such as lipofectin (Junichi etal, U.S. Pat. No. 5,705,188), cationic glycerol derivatives, andpolycationic molecules, such as polylysine (Lollo et al., PCTApplication WO 97/30731), are also known to enhance the cellular uptakeof dsRNAs.

Other agents may be utilized to enhance the penetration of theadministered nucleic acids, including glycols such as ethylene glycoland propylene glycol, pyrrols such as 2-pyrrol, azones, and terpenessuch as limonene and menthone.

Carriers

dsRNAs of the present invention can be formulated in a pharmaceuticallyacceptable carrier or diluent. A “pharmaceutically acceptable carrier”(also referred to herein as an “excipient”) is a pharmaceuticallyacceptable solvent, suspending agent, or any other pharmacologicallyinert vehicle. Pharmaceutically acceptable carriers can be liquid orsolid, and can be selected with the planned manner of administration inmind so as to provide for the desired bulk, consistency, and otherpertinent transport and chemical properties. Typical pharmaceuticallyacceptable carriers include, by way of example and not limitation:water; saline solution; binding agents (e.g., polyvinylpyrrolidone orhydroxypropyl methylcellulose); fillers (e.g., lactose and other sugars,gelatin, or calcium sulfate); lubricants (e.g., starch, polyethyleneglycol, or sodium acetate); disintegrates (e.g., starch or sodium starchglycolate); and wetting agents (e.g., sodium lauryl sulfate).

Certain compositions of the present invention also incorporate carriercompounds in the formulation. As used herein, “carrier compound” or“carrier” can refer to a nucleic acid, or analog thereof, which is inert(i.e., does not possess biological activity per se) but is recognized asa nucleic acid by in vivo processes that reduce the bioavailability of anucleic acid having biological activity by, for example, degrading thebiologically active nucleic acid or promoting its removal fromcirculation. The co-administration of a nucleic acid and a carriercompound, typically with an excess of the latter substance, can resultin a substantial reduction of the amount of nucleic acid recovered inthe liver, kidney or other extra-circulatory reservoirs, presumably dueto competition between the carrier compound and the nucleic acid for acommon receptor. For example, the recovery of a partiallyphosphorothioate dsRNA in hepatic tissue can be reduced when it isco-administered with polyinosinic acid, dextran sulfate, polycytidicacid or 4-acetamido-4′ isothiocyano-stilbene-2,2′-disulfonic acid (Miyaoet al., DsRNA Res. Dev., 1995, 5, 115-121; Takakura et al., DsRNA &Nucl. Acid Drug Dev., 1996, 6, 177-183.

Excipients

In contrast to a carrier compound, a “pharmaceutical carrier” or“excipient” is a pharmaceutically acceptable solvent, suspending agentor any other pharmacologically inert vehicle for delivering one or morenucleic acids to an animal. The excipient may be liquid or solid and isselected, with the planned manner of administration in mind, so as toprovide for the desired bulk, consistency, etc., when combined with anucleic acid and the other components of a given pharmaceuticalcomposition. Typical pharmaceutical carriers include, but are notlimited to, binding agents (e.g., pregelatinized maize starch,polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers(e.g., lactose and other sugars, microcrystalline cellulose, pectin,gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calciumhydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc,silica, colloidal silicon dioxide, stearic acid, metallic stearates,hydrogenated vegetable oils, corn starch, polyethylene glycols, sodiumbenzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodiumstarch glycolate, etc.); and wetting agents (e.g., sodium laurylsulphate, etc).

Pharmaceutically acceptable organic or inorganic excipients suitable fornon-parenteral administration which do not deleteriously react withnucleic acids can also be used to formulate the compositions of thepresent invention. Suitable pharmaceutically acceptable carriersinclude, but are not limited to, water, salt solutions, alcohols,polyethylene glycols, gelatin, lactose, amylose, magnesium stearate,talc, silicic acid, viscous paraffin, hydroxymethylcellulose,polyvinylpyrrolidone and the like.

Formulations for topical administration of nucleic acids may includesterile and non-sterile aqueous solutions, non-aqueous solutions incommon solvents such as alcohols, or solutions of the nucleic acids inliquid or solid oil bases. The solutions may also contain buffers,diluents and other suitable additives. Pharmaceutically acceptableorganic or inorganic excipients suitable for non-parenteraladministration which do not deleteriously react with nucleic acids canbe used.

Suitable pharmaceutically acceptable excipients include, but are notlimited to, water, salt solutions, alcohol, polyethylene glycols,gelatin, lactose, amylose, magnesium stearate, talc, silicic acid,viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and thelike.

Other Components

The compositions of the present invention may additionally contain otheradjunct components conventionally found in pharmaceutical compositions,at their art-established usage levels. Thus, for example, thecompositions may contain additional, compatible, pharmaceutically-activematerials such as, for example, antipruritics, astringents, localanesthetics or anti-inflammatory agents, or may contain additionalmaterials useful in physically formulating various dosage forms of thecompositions of the present invention, such as dyes, flavoring agents,preservatives, antioxidants, opacifiers, thickening agents andstabilizers. However, such materials, when added, should not undulyinterfere with the biological activities of the components of thecompositions of the present invention. The formulations can besterilized and, if desired, mixed with auxiliary agents, e.g.,lubricants, preservatives, stabilizers, wetting agents, emulsifiers,salts for influencing osmotic pressure, buffers, colorings, flavoringsand/or aromatic substances and the like which do not deleteriouslyinteract with the nucleic acid(s) of the formulation.

Aqueous suspensions may contain substances which increase the viscosityof the suspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension may also contain stabilizers.

Combination Therapy

In one aspect, a composition of the invention can be used in combinationtherapy. The term “combination therapy” includes the administration ofthe subject compounds in further combination with other biologicallyactive ingredients (such as, but not limited to, a second and differentantineoplastic agent) and non-drug therapies (such as, but not limitedto, surgery or radiation treatment). For instance, the compounds of theinvention can be used in combination with other pharmaceutically activecompounds, preferably compounds that are able to enhance the effect ofthe compounds of the invention. The compounds of the invention can beadministered simultaneously (as a single preparation or separatepreparation) or sequentially to the other drug therapy. In general, acombination therapy envisions administration of two or more drugs duringa single cycle or course of therapy.

In one aspect of the invention, the subject compounds may beadministered in combination with one or more separate agents thatmodulate protein kinases involved in various disease states. Examples ofsuch kinases may include, but are not limited to: serine/threoninespecific kinases, receptor tyrosine specific kinases and non-receptortyrosine specific kinases. Serine/threonine kinases include mitogenactivated protein kinases (MAPK), meiosis specific kinase (MEK), RAF andaurora kinase. Examples of receptor kinase families include epidermalgrowth factor receptor (EGFR) (e.g., HER2/neu, HER3, HER4, ErbB, ErbB2,ErbB3, ErbB4, Xmrk, DER, Let23); fibroblast growth factor (FGF) receptor(e.g. FGF-R1, GFF-R2/BEK/CEK3, FGF-R3/CEK2, FGF-R4/TKF, KGF-R);hepatocyte growth/scatter factor receptor (HGFR) (e.g., MET, RON, SEA,SEX); insulin receptor (e.g. IGFI-R); Eph (e.g. CEK5, CEK8, EBK, ECK,EEK, EHK-I, EHK-2, ELK, EPH, ERK, HEK, MDK2, MDK5, SEK); AxI (e.g.Mer/Nyk, Rse); RET; and platelet-derived growth factor receptor (PDGFR)(e.g. PDGFα-R, PDGβ-R, CSF1-R/FMS, SCF-R/C-KIT, VEGF-R/FLT, NEK/FLK1,FLT3/FLK2/STK-1). Non-receptor tyrosine kinase families include, but arenot limited to, BCR-ABL (e.g. p43^(abl), ARG); BTK (e.g. ITK/EMT, TEC);CSK, FAK, FPS, JAK, SRC, BMX, FER, CDK and SYK.

In another aspect of the invention, the subject compounds may beadministered in combination with one or more agents that modulatenon-kinase biological targets or processes. Such targets include histonedeacetylases (HDAC), DNA methyltransferase (DNMT), heat shock proteins(e.g., HSP90), and proteosomes.

In one embodiment, subject compounds may be combined with antineoplasticagents (e.g. small molecules, monoclonal antibodies, antisense RNA, andfusion proteins) that inhibit one or more biological targets such asZolinza, Tarceva, Iressa, Tykerb, Gleevec, Sutent, Sprycel, Nexavar,Sorafenib, CNF2024, RG108, BMS387032, Affmitak, Avastin, Herceptin,Erbitux, AG24322, PD325901, ZD6474, PD 184322, Obatodax, ABT737 andAEE788. Such combinations may enhance therapeutic efficacy over efficacyachieved by any of the agents alone and may prevent or delay theappearance of resistant mutational variants.

In certain preferred embodiments, the compounds of the invention areadministered in combination with a chemotherapeutic agent.Chemotherapeutic agents encompass a wide range of therapeutic treatmentsin the field of oncology. These agents are administered at variousstages of the disease for the purposes of shrinking tumors, destroyingremaining cancer cells left over after surgery, inducing remission,maintaining remission and/or alleviating symptoms relating to the canceror its treatment. Examples of such agents include, but are not limitedto, alkylating agents such as mustard gas derivatives (Mechlorethamine,cylophosphamide, chlorambucil, melphalan, ifosfamide), ethylenimines(thiotepa, hexamethylmelanine), Alkylsulfonates (Busulfan), Hydrazinesand Triazines (Altretamine, Procarbazine, Dacarbazine and Temozolomide),Nitrosoureas (Carmustine, Lomustine and Streptozocin), Ifosfamide andmetal salts (Carboplatin, Cisplatin, and Oxaliplatin); plant alkaloidssuch as Podophyllotoxins (Etoposide and Tenisopide), Taxanes (Paclitaxeland Docetaxel), Vinca alkaloids (Vincristine, Vinblastine, Vindesine andVinorelbine), and Camptothecan analogs (Irinotecan and Topotecan);anti-tumor antibiotics such as Chromomycins (Dactinomycin andPlicamycin), Anthracyclines (Doxorubicin, Daunorubicin, Epirubicin,Mitoxantrone, Valrubicin and Idarubicin), and miscellaneous antibioticssuch as Mitomycin, Actinomycin and Bleomycin; anti-metabolites such asfolic acid antagonists (Methotrexate, Pemetrexed, Raltitrexed,Aminopterin), pyrimidine antagonists (5-Fluorouracil, Floxuridine,Cytarabine, Capecitabine, and Gemcitabine), purine antagonists(6-Mercaptopurine and 6-Thioguanine) and adenosine deaminase inhibitors(Cladribine, Fludarabine, Mercaptopurine, Clofarabine, Thioguanine,Nelarabine and Pentostatin); topoisomerase inhibitors such astopoisomerase I inhibitors (Ironotecan, topotecan) and topoisomerase IIinhibitors (Amsacrine, etoposide, etoposide phosphate, teniposide);monoclonal antibodies (Alemtuzumab, Gemtuzumab ozogamicin, Rituximab,Trastuzumab, Ibritumomab Tioxetan, Cetuximab, Panitumumab, Tositumomab,Bevacizumab); and miscellaneous anti-neoplasties such as ribonucleotidereductase inhibitors (Hydroxyurea); adrenocortical steroid inhibitor(Mitotane); enzymes (Asparaginase and Pegaspargase); anti-microtubuleagents (Estramustine); and retinoids (Bexarotene, Isotretinoin,Tretinoin (ATRA). In certain preferred embodiments, the compounds of theinvention are administered in combination with a chemoprotective agent.Chemoprotective agents act to protect the body or minimize the sideeffects of chemotherapy. Examples of such agents include, but are notlimited to, amfostine, mesna, and dexrazoxane.

In one aspect of the invention, the subject compounds are administeredin combination with radiation therapy. Radiation is commonly deliveredinternally (implantation of radioactive material near cancer site) orexternally from a machine that employs photon (x-ray or gamma-ray) orparticle radiation. Where the combination therapy further comprisesradiation treatment, the radiation treatment may be conducted at anysuitable time so long as a beneficial effect from the co-action of thecombination of the therapeutic agents and radiation treatment isachieved. For example, in appropriate cases, the beneficial effect isstill achieved when the radiation treatment is temporally removed fromthe administration of the therapeutic agents, perhaps by days or evenweeks.

It will be appreciated that compounds of the invention can be used incombination with an immunotherapeutic agent. One form of immunotherapyis the generation of an active systemic tumor-specific immune responseof host origin by administering a vaccine composition at a site distantfrom the tumor. Various types of vaccines have been proposed, includingisolated tumor-antigen vaccines and anti-idiotype vaccines. Anotherapproach is to use tumor cells from the subject to be treated, or aderivative of such cells (reviewed by Schirrmacher et al. (1995) J.Cancer Res. Clin. Oncol. 121:487). In U.S. Pat. No. 5,484,596, Hanna Jr.et al. claim a method for treating a resectable carcinoma to preventrecurrence or metastases, comprising surgically removing the tumor,dispersing the cells with collagenase, irradiating the cells, andvaccinating the patient with at least three consecutive doses of about10⁷ cells.

It will be appreciated that the compounds of the invention mayadvantageously be used in conjunction with one or more adjunctivetherapeutic agents. Examples of suitable agents for adjunctive therapyinclude steroids, such as corticosteroids (amcinonide, betamethasone,betamethasone dipropionate, betamethasone valerate, budesonide,clobetasol, clobetasol acetate, clobetasol butyrate, clobetasol17-propionate, cortisone, deflazacort, desoximetasone, diflucortolonevalerate, dexamethasone, dexamethasone sodium phosphate, desonide,furoate, fluocinonide, fluocinolone acetonide, halcinonide,hydrocortisone, hydrocortisone butyrate, hydrocortisone sodiumsuccinate, hydrocortisone valerate, methyl prednisolone, mometasone,prednicarbate, prednisolone, triamcinolone, triamcinolone acetonide, andhalobetasol proprionate); a 5HTi agonist, such as a triptan (e.g.sumatriptan or naratriptan); an adenosine A1 agonist; an EP ligand; anNMDA modulator, such as a glycine antagonist; a sodium channel blocker(e.g. lamotrigine); a substance P antagonist (e.g. an NKi antagonist); acannabinoid; acetaminophen or phenacetin; a 5-lipoxygenase inhibitor; aleukotriene receptor antagonist; a DMARD (e.g. methotrexate); gabapentinand related compounds; a tricyclic antidepressant (e.g. amitryptilline);a neurone stabilizing antiepileptic drug; a mono-aminergic uptakeinhibitor (e.g. venlafaxine); a matrix metalloproteinase inhibitor; anitric oxide synthase (NOS) inhibitor, such as an iNOS or an nNOSinhibitor; an inhibitor of the release, or action, of tumour necrosisfactor α; an antibody therapy, such as a monoclonal antibody therapy; anantiviral agent, such as a nucleoside inhibitor (e.g. lamivudine) or animmune system modulator (e.g. interferon); an opioid analgesic; a localanaesthetic; a stimulant, including caffeine; an H2-antagonist (e.g.ranitidine); a proton pump inhibitor (e.g. omeprazole); an antacid (e.g.aluminium or magnesium hydroxide; an antiflatulent (e.g. simethicone); adecongestant (e.g. phenylephrine, phenylpropanolamine, pseudoephedrine,oxymetazoline, epinephrine, naphazoline, xylometazoline,propylhexedrine, or levo-desoxyephedrine); an antitussive (e.g. codeine,hydrocodone, carmiphen, carbetapentane, or dextramethorphan); adiuretic; or a sedating or non-sedating antihistamine.

The compounds of the invention can be co-administered with siRNA thattarget other genes. For example, a compound of the invention can beco-administered with an siRNA targeted to a c-Myc gene. In one example,AD-12115 can be co-administered with a c-Myc siRNA. Examples of c-Myctargeted siRNAs are disclosed in U.S. patent application Ser. No.12/373,039 which is herein incorporated by reference.

Methods of Preparing Lipid Particles

The methods and compositions of the invention make use of certaincationic lipids, the synthesis, preparation and characterization ofwhich is described below and in the accompanying Examples. In addition,the present invention provides methods of preparing lipid particles,including those associated with a therapeutic agent, e.g., a nucleicacid. In the methods described herein, a mixture of lipids is combinedwith a buffered aqueous solution of nucleic acid to produce anintermediate mixture containing nucleic acid encapsulated in lipidparticles wherein the encapsulated nucleic acids are present in anucleic acid/lipid ratio of about 3 wt % to about 25 wt %, preferably 5to 15 wt %. The intermediate mixture may optionally be sized to obtainlipid-encapsulated nucleic acid particles wherein the lipid portions areunilamellar vesicles, preferably having a diameter of 30 to 150 nm, morepreferably about 40 to 90 nm. The pH is then raised to neutralize atleast a portion of the surface charges on the lipid-nucleic acidparticles, thus providing an at least partially surface-neutralizedlipid-encapsulated nucleic acid composition.

As described above, several of these cationic lipids are amino lipidsthat are charged at a pH below the pK_(a) of the amino group andsubstantially neutral at a pH above the pK_(a). These cationic lipidsare termed titratable cationic lipids and can be used in theformulations of the invention using a two-step process. First, lipidvesicles can be formed at the lower pH with titratable cationic lipidsand other vesicle components in the presence of nucleic acids. In thismanner, the vesicles will encapsulate and entrap the nucleic acids.Second, the surface charge of the newly formed vesicles can beneutralized by increasing the pH of the medium to a level above thepK_(a) of the titratable cationic lipids present, i.e., to physiologicalpH or higher. Particularly advantageous aspects of this process includeboth the facile removal of any surface adsorbed nucleic acid and aresultant nucleic acid delivery vehicle which has a neutral surface.Liposomes or lipid particles having a neutral surface are expected toavoid rapid clearance from circulation and to avoid certain toxicitieswhich are associated with cationic liposome preparations. Additionaldetails concerning these uses of such titratable cationic lipids in theformulation of nucleic acid-lipid particles are provided in U.S. Pat.No. 6,287,591 and U.S. Pat. No. 6,858,225, incorporated herein byreference.

It is further noted that the vesicles formed in this manner provideformulations of uniform vesicle size with high content of nucleic acids.Additionally, the vesicles have a size range of from about 30 to about150 nm, more preferably about 30 to about 90 nm.

Without intending to be bound by any particular theory, it is believedthat the very high efficiency of nucleic acid encapsulation is a resultof electrostatic interaction at low pH. At acidic pH (e.g. pH 4.0) thevesicle surface is charged and binds a portion of the nucleic acidsthrough electrostatic interactions. When the external acidic buffer isexchanged for a more neutral buffer (e.g. pH 7.5) the surface of thelipid particle or liposome is neutralized, allowing any external nucleicacid to be removed. More detailed information on the formulation processis provided in various publications (e.g., U.S. Pat. No. 6,287,591 andU.S. Pat. No. 6,858,225).

In view of the above, the present invention provides methods ofpreparing lipid/nucleic acid formulations. In the methods describedherein, a mixture of lipids is combined with a buffered aqueous solutionof nucleic acid to produce an intermediate mixture containing nucleicacid encapsulated in lipid particles, e.g., wherein the encapsulatednucleic acids are present in a nucleic acid/lipid ratio of about 10 wt %to about 20 wt %. The intermediate mixture may optionally be sized toobtain lipid-encapsulated nucleic acid particles wherein the lipidportions are unilamellar vesicles, preferably having a diameter of 30 to150 nm, more preferably about 40 to 90 nm. The pH is then raised toneutralize at least a portion of the surface charges on thelipid-nucleic acid particles, thus providing an at least partiallysurface-neutralized lipid-encapsulated nucleic acid composition.

In certain embodiments, the mixture of lipids includes at least twolipid components: a first amino lipid component of the present inventionthat is selected from among lipids which have a pKa such that the lipidis cationic at pH below the pKa and neutral at pH above the pKa, and asecond lipid component that is selected from among lipids that preventparticle aggregation during lipid-nucleic acid particle formation. Inparticular embodiments, the amino lipid is a novel cationic lipid of thepresent invention.

In preparing the nucleic acid-lipid particles of the invention, themixture of lipids is typically a solution of lipids in an organicsolvent. This mixture of lipids can then be dried to form a thin film orlyophilized to form a powder before being hydrated with an aqueousbuffer to form liposomes. Alternatively, in a preferred method, thelipid mixture can be solubilized in a water miscible alcohol, such asethanol, and this ethanolic solution added to an aqueous bufferresulting in spontaneous liposome formation. In most embodiments, thealcohol is used in the form in which it is commercially available. Forexample, ethanol can be used as absolute ethanol (100%), or as 95%ethanol, the remainder being water. This method is described in moredetail in U.S. Pat. No. 5,976,567).

In accordance with the invention, the lipid mixture is combined with abuffered aqueous solution that may contain the nucleic acids. Thebuffered aqueous solution of is typically a solution in which the bufferhas a pH of less than the pK_(a) of the protonatable lipid in the lipidmixture. Examples of suitable buffers include citrate, phosphate,acetate, and MES. A particularly preferred buffer is citrate buffer.Preferred buffers will be in the range of 1-1000 mM of the anion,depending on the chemistry of the nucleic acid being encapsulated, andoptimization of buffer concentration may be significant to achievinghigh loading levels (see, e.g., U.S. Pat. No. 6,287,591 and U.S. Pat.No. 6,858,225). Alternatively, pure water acidified to pH 5-6 withchloride, sulfate or the like may be useful. In this case, it may besuitable to add 5% glucose, or another non-ionic solute which willbalance the osmotic potential across the particle membrane when theparticles are dialyzed to remove ethanol, increase the pH, or mixed witha pharmaceutically acceptable carrier such as normal saline. The amountof nucleic acid in buffer can vary, but will typically be from about0.01 mg/mL to about 200 mg/mL, more preferably from about 0.5 mg/mL toabout 50 mg/mL.

The mixture of lipids and the buffered aqueous solution of therapeuticnucleic acids is combined to provide an intermediate mixture. Theintermediate mixture is typically a mixture of lipid particles havingencapsulated nucleic acids. Additionally, the intermediate mixture mayalso contain some portion of nucleic acids which are attached to thesurface of the lipid particles (liposomes or lipid vesicles) due to theionic attraction of the negatively-charged nucleic acids andpositively-charged lipids on the lipid particle surface (the aminolipids or other lipid making up the protonatable first lipid componentare positively charged in a buffer having a pH of less than the pK_(a)of the protonatable group on the lipid). In one group of preferredembodiments, the mixture of lipids is an alcohol solution of lipids andthe volumes of each of the solutions are adjusted so that uponcombination, the resulting alcohol content is from about 20% by volumeto about 45% by volume. The method of combining the mixtures can includeany of a variety of processes, often depending upon the scale offormulation produced. For example, when the total volume is about 10-20mL or less, the solutions can be combined in a test tube and stirredtogether using a vortex mixer. Large-scale processes can be carried outin suitable production scale glassware.

Optionally, the lipid-encapsulated therapeutic agent (e.g., nucleicacid) complexes which are produced by combining the lipid mixture andthe buffered aqueous solution of therapeutic agents (nucleic acids) canbe sized to achieve a desired size range and relatively narrowdistribution of lipid particle sizes. Preferably, the compositionsprovided herein will be sized to a mean diameter of from about 70 toabout 200 nm, more preferably about 90 to about 130 nm. Severaltechniques are available for sizing liposomes to a desired size. Onesizing method is described in U.S. Pat. No. 4,737,323, incorporatedherein by reference. Sonicating a liposome suspension either by bath orprobe sonication produces a progressive size reduction down to smallunilamellar vesicles (SUVs) less than about 0.05 microns in size.Homogenization is another method which relies on shearing energy tofragment large liposomes into smaller ones. In a typical homogenizationprocedure, multilamellar vesicles are recirculated through a standardemulsion homogenizer until selected liposome sizes, typically betweenabout 0.1 and 0.5 microns, are observed. In both methods, the particlesize distribution can be monitored by conventional laser-beam particlesize determination. For certain methods herein, extrusion is used toobtain a uniform vesicle size.

Extrusion of liposome compositions through a small-pore polycarbonatemembrane or an asymmetric ceramic membrane results in a relativelywell-defined size distribution. Typically, the suspension is cycledthrough the membrane one or more times until the desired liposomecomplex size distribution is achieved. The liposomes may be extrudedthrough successively smaller-pore membranes, to achieve a gradualreduction in liposome size. In some instances, the lipid-nucleic acidcompositions which are formed can be used without any sizing.

In particular embodiments, methods of the present invention furthercomprise a step of neutralizing at least some of the surface charges onthe lipid portions of the lipid-nucleic acid compositions. By at leastpartially neutralizing the surface charges, unencapsulated nucleic acidis freed from the lipid particle surface and can be removed from thecomposition using conventional techniques. Preferably, unencapsulatedand surface adsorbed nucleic acids are removed from the resultingcompositions through exchange of buffer solutions. For example,replacement of a citrate buffer (pH about 4.0, used for forming thecompositions) with a HEPES-buffered saline (HBS pH about 7.5) solution,results in the neutralization of liposome surface and nucleic acidrelease from the surface. The released nucleic acid can then be removedvia chromatography using standard methods, and then switched into abuffer with a pH above the pKa of the lipid used.

Optionally the lipid vesicles (i.e., lipid particles) can be formed byhydration in an aqueous buffer and sized using any of the methodsdescribed above prior to addition of the nucleic acid. As describedabove, the aqueous buffer should be of a pH below the pKa of the aminolipid. A solution of the nucleic acids can then be added to these sized,preformed vesicles. To allow encapsulation of nucleic acids into such“pre-formed” vesicles the mixture should contain an alcohol, such asethanol. In the case of ethanol, it should be present at a concentrationof about 20% (w/w) to about 45% (w/w). In addition, it may be necessaryto warm the mixture of pre-formed vesicles and nucleic acid in theaqueous buffer-ethanol mixture to a temperature of about 25° C. to about50° C. depending on the composition of the lipid vesicles and the natureof the nucleic acid. It will be apparent to one of ordinary skill in theart that optimization of the encapsulation process to achieve a desiredlevel of nucleic acid in the lipid vesicles will require manipulation ofvariable such as ethanol concentration and temperature. Examples ofsuitable conditions for nucleic acid encapsulation are provided in theExamples. Once the nucleic acids are encapsulated within the preformedvesicles, the external pH can be increased to at least partiallyneutralize the surface charge. Unencapsulated and surface adsorbednucleic acids can then be removed as described above.

Methods for Inhibiting Expression of the PCSK9 Gene

In yet another aspect, the invention provides a method for inhibitingthe expression of the PCSK9 gene in a mammal. The method includesadministering a composition of the invention to the mammal such thatexpression of the target PCSK9 gene is decreased for an extendedduration, e.g., at least one week, two weeks, three weeks, or four weeksor longer.

For example, in certain instances, expression of the PCSK9 gene issuppressed by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,or 50% by administration of a double-stranded oligonucleotide describedherein. In some embodiments, the PCSK9 gene is suppressed by at leastabout 60%, 70%, or 80% by administration of the double-strandedoligonucleotide. In some embodiments, the PCSK9 gene is suppressed by atleast about 85%, 90%, or 95% by administration of the double-strandedoligonucleotide. Table 1b, Table 2b, and Table 5b provide a wide rangeof values for inhibition of expression obtained in an in vitro assayusing various PCSK9 dsRNA molecules at various concentrations.

The effect of the decreased target PCSK9 gene preferably results in adecrease in LDLc (low density lipoprotein cholesterol) levels in theblood, and more particularly in the serum, of the mammal. In someembodiments, LDLc levels are decreased by at least 10%, 15%, 20%, 25%,30%, 40%, 50%, or 60%, or more, as compared to pretreatment levels.

The method includes administering a composition containing a dsRNA,where the dsRNA has a nucleotide sequence that is complementary to atleast a part of an RNA transcript of the PCSK9 gene of the mammal to betreated. When the organism to be treated is a mammal such as a human,the composition can be administered by any means known in the artincluding, but not limited to oral or parenteral routes, includingintravenous, intramuscular, subcutaneous, transdermal, and airway(aerosol) administration. In some embodiments, the compositions areadministered by intravenous infusion or injection.

The methods and compositions described herein can be used to treatdiseases and conditions that can be modulated by down regulating PCSK9gene expression. For example, the compositions described herein can beused to treat hyperlipidemia and other forms of lipid imbalance such ashypercholesterolemia, hypertriglyceridemia and the pathologicalconditions associated with these disorders such as heart and circulatorydiseases. In some embodiments, a patient treated with a PCSK9 dsRNA isalso administered a non-dsRNA therapeutic agent, such as an agent knownto treat lipid disorders.

In one aspect, the invention provides a method of inhibiting theexpression of the PCSK9 gene in a subject, e.g., a human. The methodincludes administering a first single dose of dsRNA, e.g., a dosesufficient to depress levels of PCSK9 mRNA for at least 5, morepreferably 7, 10, 14, 21, 25, 30 or 40 days; and optionally,administering a second single dose of dsRNA, wherein the second singledose is administered at least 5, more preferably 7, 10, 14, 21, 25, 30or 40 days after the first single dose is administered, therebyinhibiting the expression of the PCSK9 gene in a subject.

In one embodiment, doses of dsRNA are administered not more than onceevery four weeks, not more than once every three weeks, not more thanonce every two weeks, or not more than once every week. In anotherembodiment, the administrations can be maintained for one, two, three,or six months, or one year or longer.

In another embodiment, administration can be provided when Low DensityLipoprotein cholesterol (LDLc) levels reach or surpass a predeterminedminimal level, such as greater than 70 mg/dL, 130 mg/dL, 150 mg/dL, 200mg/dL, 300 mg/dL, or 400 mg/dL.

In one embodiment, the subject is selected, at least in part, on thebasis of needing (as opposed to merely selecting a patient on thegrounds of who happens to be in need of) LDL lowering, LDL loweringwithout lowering of HDL, ApoB lowering, or total cholesterol loweringwithout HDL lowering.

In one embodiment, the dsRNA does not activate the immune system, e.g.,it does not increase cytokine levels, such as TNF-alpha or IFN-alphalevels. For example, when measured by an assay, such as an in vitro PBMCassay, such as described herein, the increase in levels of TNF-alpha orIFN-alpha, is less than 30%, 20%, or 10% of control cells treated with acontrol dsRNA, such as a dsRNA that does not target PCSK9.

In one aspect, the invention provides a method for treating, preventingor managing a disorder, pathological process or symptom, which, forexample, can be mediated by down regulating PCSK9 gene expression in asubject, such as a human subject. In one embodiment, the disorder ishyperlipidemia. The method includes administering a first single dose ofdsRNA, e.g., a dose sufficient to depress levels of PCSK9 mRNA for atleast 5, more preferably 7, 10, 14, 21, 25, 30 or 40 days; andoptionally, administering a second single dose of dsRNA, wherein thesecond single dose is administered at least 5, more preferably 7, 10,14, 21, 25, 30 or 40 days after the first single dose is administered,thereby inhibiting the expression of the PCSK9 gene in a subject.

In another embodiment, a composition containing a dsRNA featured in theinvention, i.e., a dsRNA targeting PCSK9, is administered with anon-dsRNA therapeutic agent, such as an agent known to treat a lipiddisorders, such as hypercholesterolemia, atherosclerosis ordyslipidemia. For example, a dsRNA featured in the invention can beadministered with, e.g., an HMG-CoA reductase inhibitor (e.g., astatin), a fibrate, a bile acid sequestrant, niacin, an antiplateletagent, an angiotensin converting enzyme inhibitor, an angiotensin IIreceptor antagonist (e.g., losartan potassium, such as Merck & Co.'sCozaar®), an acylCoA cholesterol acetyltransferase (ACAT) inhibitor, acholesterol absorption inhibitor, a cholesterol ester transfer protein(CETP) inhibitor, a microsomal triglyceride transfer protein (MTTP)inhibitor, a cholesterol modulator, a bile acid modulator, a peroxisomeproliferation activated receptor (PPAR) agonist, a gene-based therapy, acomposite vascular protectant (e.g., AGI-1067, from Atherogenics), aglycoprotein IIb/IIIa inhibitor, aspirin or an aspirin-like compound, anIBAT inhibitor (e.g., S-8921, from Shionogi), a squalene synthaseinhibitor, or a monocyte chemoattractant protein (MCP)-I inhibitor.Exemplary HMG-CoA reductase inhibitors include atorvastatin (Pfizer'sLipitor®/Tahor/Sortis/Torvast/Cardyl), pravastatin (Bristol-MyersSquibb's Pravachol, Sankyo's Mevalotin/Sanaprav), simvastatin (Merck'sZocor®/Sinvacor, Boehringer Ingelheim's Denan, Banyu's Lipovas),lovastatin (Merck's Mevacor/Mevinacor, Bexal's Lovastatina, Cepa;Schwarz Pharma's Liposcler), fluvastatin (Novartis'Lescol®/Locol/Lochol, Fujisawa's Cranoc, Solvay's Digaril), cerivastatin(Bayer's Lipobay/GlaxoSmithKline's Baycol), rosuvastatin (AstraZeneca'sCrestor®), and pitivastatin (itavastatin/risivastatin) (Nissan Chemical,Kowa Kogyo, Sankyo, and Novartis). Exemplary fibrates include, e.g.,bezafibrate (e.g., Roche's Befizal®/Cedur®/Bezalip®, Kissei's Bezatol),clofibrate (e.g., Wyeth's Atromid-S®), fenofibrate (e.g., Fournier'sLipidil/Lipantil, Abbott's Tricor®, Takeda's Lipantil, generics),gemfibrozil (e.g., Pfizer's Lopid/Lipur) and ciprofibrate(Sanofi-Synthelabo's Modalim®). Exemplary bile acid sequestrantsinclude, e.g., cholestyramine (Bristol-Myers Squibb's Questran® andQuestran Light™), colestipol (e.g., Pharmacia's Colestid), andcolesevelam (Genzyme/Sankyo's WelChol™). Exemplary niacin therapiesinclude, e.g., immediate release formulations, such as Aventis' Nicobid,Upsher-Smith's Niacor, Aventis' Nicolar, and Sanwakagaku's Perycit.Niacin extended release formulations include, e.g., Kos Pharmaceuticals'Niaspan and Upsher-Smith's SIo-Niacin. Exemplary antiplatelet agentsinclude, e.g., aspirin (e.g., Bayer's aspirin), clopidogrel(Sanofi-Synthelabo/Bristol-Myers Squibb's Plavix), and ticlopidine(e.g., Sanofi-Synthelabo's Ticlid and Daiichi's Panaldine). Otheraspirin-like compounds useful in combination with a dsRNA targetingPCSK9 include, e.g., Asacard (slow-release aspirin, by Pharmacia) andPamicogrel (Kanebo/Angelini Ricerche/CEPA). Exemplaryangiotensin-converting enzyme inhibitors include, e.g., ramipril (e.g.,Aventis' Altace) and enalapril (e.g., Merck & Co.'s Vasotec). Exemplaryacyl CoA cholesterol acetyltransferase (ACAT) inhibitors include, e.g.,avasimibe (Pfizer), eflucimibe (BioM{acute over (ε)}rieux PierreFabre/Eli Lilly), CS-505 (Sankyo and Kyoto), and SMP-797 (Sumito).Exemplary cholesterol absorption inhibitors include, e.g., ezetimibe(Merck/Schering-Plough Pharmaceuticals Zetia®) and Pamaqueside (Pfizer).Exemplary CETP inhibitors include, e.g., Torcetrapib (also calledCP-529414, Pfizer), JTT-705 (Japan Tobacco), and CETi-I (AvantImmunotherapeutics). Exemplary microsomal triglyceride transfer protein(MTTP) inhibitors include, e.g., implitapide (Bayer), R-103757(Janssen), and CP-346086 (Pfizer). Other exemplary cholesterolmodulators include, e.g., NO-1886 (Otsuka/TAP Pharmaceutical), CI-1027(Pfizer), and WAY-135433 (Wyeth-Ayerst). Exemplary bile acid modulatorsinclude, e.g., HBS-107 (Hisamitsu/Banyu), Btg-511 (British TechnologyGroup), BARI-1453 (Aventis), S-8921 (Shionogi), SD-5613 (Pfizer), andAZD-7806 (AstraZeneca). Exemplary peroxisome proliferation activatedreceptor (PPAR) agonists include, e.g., tesaglitazar (AZ-242)(AstraZeneca), Netoglitazone (MCC-555) (Mitsubishi/Johnson & Johnson),GW-409544 (Ligand Pharmaceuticals/GlaxoSmithKline), GW-501516 (LigandPharmaceuticals/GlaxoSmithKline), LY-929 (Ligand Pharmaceuticals and EliLilly), LY-465608 (Ligand Pharmaceuticals and Eli Lilly), LY-518674(Ligand Pharmaceuticals and Eli Lilly), and MK-767 (Merck and Kyorin).Exemplary gene-based therapies include, e.g., AdGWEGF121.10 (GenVec),ApoA1 (UCB Pharma/Groupe Fournier), EG-004 (Trinam) (Ark Therapeutics),and ATP-binding cassette transporter-A1 (ABCA1) (CV Therapeutics/Incyte,Aventis, Xenon). Exemplary Glycoprotein Ilb/IIIa inhibitors include,e.g., roxifiban (also called DMP754, Bristol-Myers Squibb), Gantofiban(Merck KGaA/Yamanouchi), and Cromafiban (Millennium Pharmaceuticals).Exemplary squalene synthase inhibitors include, e.g., BMS-1884941(Bristol-Myers Squibb), CP-210172 (Pfizer), CP-295697 (Pfizer),CP-294838 (Pfizer), and TAK-475 (Takeda). An exemplary MCP-I inhibitoris, e.g., RS-504393 (Roche Bioscience). The anti-atherosclerotic agentBO-653 (Chugai Pharmaceuticals), and the nicotinic acid derivativeNyclin (Yamanouchi Pharmaceuticals) are also appropriate foradministering in combination with a dsRNA featured in the invention.Exemplary combination therapies suitable for administration with a dsRNAtargeting PCSK9 include, e.g., advicor (Niacin/lovastatin from KosPharmaceuticals), amlodipine/atorvastatin (Pfizer), andezetimibe/simvastatin (e.g., Vytorin® 10/10, 10/20, 10/40, and 10/80tablets by Merck/Schering-Plough Pharmaceuticals). Agents for treatinghypercholesterolemia, and suitable for administration in combinationwith a dsRNA targeting PCSK9 include, e.g., lovastatin, niacin Altoprev®Extended-Release Tablets (Andrx Labs), lovastatin Caduet® Tablets(Pfizer), amlodipine besylate, atorvastatin calcium Crestor® Tablets(AstraZeneca), rosuvastatin calcium Lescol® Capsules (Novartis),fluvastatin sodium Lescol® (Reliant, Novartis), fluvastatin sodiumLipitor® Tablets (Parke-Davis), atorvastatin calcium Lofibra® Capsules(Gate), Niaspan Extended-Release Tablets (Kos), niacin Pravachol Tablets(Bristol-Myers Squibb), pravastatin sodium TriCor® Tablets (Abbott),fenofibrate Vytorin® 10/10 Tablets (Merck/Schering-PloughPharmaceuticals), ezetimibe, simvastatin WelChol™ Tablets (Sankyo),colesevelam hydrochloride Zetia® Tablets (Schering), ezetimibe Zetia®Tablets (Merck/Schering-Plough Pharmaceuticals), and ezetimibe Zocor®Tablets (Merck).

In one embodiment, a dsRNA targeting PCSK9 is administered incombination with an ezetimibe/simvastatin combination (e.g., Vytorin®(Merck/Schering-Plough Pharmaceuticals)).

In one embodiment, the PCSK9 dsRNA is administered to the patient, andthen the non-dsRNA agent is administered to the patient (or vice versa).In another embodiment, the PCSK9 dsRNA and the non-dsRNA therapeuticagent are administered at the same time.

In another aspect, the invention features, a method of instructing anend user, e.g., a caregiver or a subject, on how to administer a dsRNAdescribed herein. The method includes, optionally, providing the enduser with one or more doses of the dsRNA, and instructing the end userto administer the dsRNA on a regimen described herein, therebyinstructing the end user.

In yet another aspect, the invention provides a method of treating apatient by selecting a patient on the basis that the patient is in needof LDL lowering, LDL lowering without lowering of HDL, ApoB lowering, ortotal cholesterol lowering. The method includes administering to thepatient a dsRNA targeting PCSK9 in an amount sufficient to lower thepatient's LDL levels or ApoB levels, e.g., without substantiallylowering HDL levels.

In another aspect, the invention provides a method of treating a patientby selecting a patient on the basis that the patient is in need oflowered ApoB levels, and administering to the patient a dsRNA targetingPCSK9 in an amount sufficient to lower the patient's ApoB levels. In oneembodiment, the amount of PCSK9 is sufficient to lower LDL levels aswell as ApoB levels. In another embodiment, administration of the PCSK9dsRNA does not affect the level of HDL cholesterol in the patient.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the invention, suitable methods and materials aredescribed below. All publications, patent applications, patents, andother references mentioned herein are incorporated by reference in theirentirety. In case of conflict, the present specification, includingdefinitions, will control. In addition, the materials, methods, andexamples are illustrative only and not intended to be limiting.

EXAMPLES Example 1 Gene Walking of the PCSK9 Gene

siRNA design was carried out to identify in two separate selections

a) siRNAs targeting PCSK9 human and either mouse or rat mRNA and

b) all human reactive siRNAs with predicted specificity to the targetgene PCSK9.

mRNA sequences to human, mouse and rat PCSK9 were used: Human sequenceNM_(—)174936.2 was used as reference sequence during the complete siRNAselection procedure.

19 mer stretches conserved in human and mouse, and human and rat PCSK9mRNA sequences were identified in the first step, resulting in theselection of siRNAs cross-reactive to human and mouse, and siRNAscross-reactive to human and rat targets

SiRNAs specifically targeting human PCSK9 were identified in a secondselection. All potential 19mer sequences of human PCSK9 were extractedand defined as candidate target sequences. Sequences cross-reactive tohuman, monkey, and those cross-reactive to mouse, rat, human and monkeyare all listed in Tables 1a and 2a. Chemically modified versions ofthose sequences and their activity in both in vitro and in vivo assaysare also listed in Tables 1a and 2a. The data is described in theexamples and in FIGS. 2-8.

In order to rank candidate target sequences and their correspondingsiRNAs and select appropriate ones, their predicted potential forinteracting with irrelevant targets (off-target potential) was taken asa ranking parameter. siRNAs with low off-target potential were definedas preferable and assumed to be more specific in vivo.

For predicting siRNA-specific off-target potential, the followingassumptions were made:

1) positions 2 to 9 (counting 5′ to 3′) of a strand (seed region) maycontribute more to off-target potential than rest of sequence (non-seedand cleavage site region)

2) positions 10 and 11 (counting 5′ to 3′) of a strand (cleavage siteregion) may contribute more to off-target potential than non-seed region

3) positions 1 and 19 of each strand are not relevant for off-targetinteractions

4) an off-target score can be calculated for each gene and each strand,based on complementarity of siRNA strand sequence to the gene's sequenceand position of mismatches

5) number of predicted off-targets as well as highest off-target scoremust be considered for off-target potential

6) off-target scores are to be considered more relevant for off-targetpotential than numbers of off-targets

7) assuming potential abortion of sense strand activity by internalmodifications introduced, only off-target potential of antisense strandwill be relevant To identify potential off-target genes, 19mer candidatesequences were subjected to a homology search against publicallyavailable human mRNA sequences.

The following off-target properties for each 19mer input sequence wereextracted for each off-target gene to calculate the off-target score:

Number of mismatches in non-seed region

Number of mismatches in seed region

Number of mismatches in cleavage site region

The off-target score was calculated for considering assumption 1 to 3 asfollows:

Off-target score=number of seed mismatches*10+number of cleavage sitemismatches*1.2+number of non-seed mismatches*1

The most relevant off-target gene for each siRNA corresponding to theinput 19mer sequence was defined as the gene with the lowest off-targetscore. Accordingly, the lowest off-target score was defined as therelevant off-target score for each siRNA.

Example 2 dsRNA Synthesis

Source of Reagents

Where the source of a reagent is not specifically given herein, suchreagent may be obtained from any supplier of reagents for molecularbiology at a quality/purity standard for application in molecularbiology.

siRNA Synthesis

Single-stranded RNAs were produced by solid phase synthesis on a scaleof 1 μmole using an Expedite 8909 synthesizer (Applied Biosystems,Applera Deutschland GmbH, Darmstadt, Germany) and controlled pore glass(CPG, 500 Å, Proligo Biochemie GmbH, Hamburg, Germany) as solid support.RNA and RNA containing 2′-O-methyl nucleotides were generated by solidphase synthesis employing the corresponding phosphoramidites and2′-O-methyl phosphoramidites, respectively (Proligo Biochemie GmbH,Hamburg, Germany). These building blocks were incorporated at selectedsites within the sequence of the oligoribonucleotide chain usingstandard nucleoside phosphoramidite chemistry such as described inCurrent protocols in nucleic acid chemistry, Beaucage, S. L. et al.(Edrs.), John Wiley & Sons, Inc., New York, N.Y., USA. Phosphorothioatelinkages were introduced by replacement of the iodine oxidizer solutionwith a solution of the Beaucage reagent (Chruachem Ltd, Glasgow, UK) inacetonitrile (1%). Further ancillary reagents were obtained fromMallinckrodt Baker (Griesheim, Germany).

Deprotection and purification of the crude oligoribonucleotides by anionexchange HPLC were carried out according to established procedures.Yields and concentrations were determined by UV absorption of a solutionof the respective RNA at a wavelength of 260 nm using a spectralphotometer (DU 640B, Beckman Coulter GmbH, Unterschlei

heim, Germany). Double stranded RNA was generated by mixing an equimolarsolution of complementary strands in annealing buffer (20 mM sodiumphosphate, pH 6.8; 100 mM sodium chloride), heated in a water bath at85-90° C. for 3 minutes and cooled to room temperature over a period of3-4 hours. The annealed RNA solution was stored at −20° C. until use.

Conjugated siRNAs

For the synthesis of 3′-cholesterol-conjugated siRNAs (herein referredto as -Chol-3), an appropriately modified solid support was used for RNAsynthesis. The modified solid support was prepared as follows:

Diethyl-2-azabutane-1,4-dicarboxylate AA

A 4.7 M aqueous solution of sodium hydroxide (50 ml) was added into astirred, ice-cooled solution of ethyl glycinate hydrochloride (32.19 g,0.23 mole) in water (50 ml). Then, ethyl acrylate (23.1 g, 0.23 mole)was added and the mixture was stirred at room temperature untilcompletion of the reaction was ascertained by TLC. After 19 h thesolution was partitioned with dichloromethane (3×100 ml). The organiclayer was dried with anhydrous sodium sulfate, filtered and evaporated.The residue was distilled to afford AA (28.8 g, 61%).

3-{Ethoxycarbonylmethyl-[6-(9H-fluoren-9-ylmethoxycarbonyl-amino)-hexanoyl]-amino}-propionicacid ethyl ester AB

Fmoc-6-amino-hexanoic acid (9.12 g, 25.83 mmol) was dissolved indichloromethane (50 ml) and cooled with ice. Diisopropylcarbodiimde(3.25 g, 3.99 ml, 25.83 mmol) was added to the solution at 0° C. It wasthen followed by the addition of Diethyl-azabutane-1,4-dicarboxylate (5g, 24.6 mmol) and dimethylamino pyridine (0.305 g, 2.5 mmol). Thesolution was brought to room temperature and stirred further for 6 h.Completion of the reaction was ascertained by TLC. The reaction mixturewas concentrated under vacuum and ethyl acetate was added to precipitatediisopropyl urea. The suspension was filtered. The filtrate was washedwith 5% aqueous hydrochloric acid, 5% sodium bicarbonate and water. Thecombined organic layer was dried over sodium sulfate and concentrated togive the crude product which was purified by column chromatography (50%EtOAC/Hexanes) to yield 11.87 g (88%) of AB.

3-[(6-Amino-hexanoyl)-ethoxycarbonylmethyl-amino]-propionic acid ethylester AC

3-{Ethoxycarbonylmethyl-[6-(9H-fluoren-9-ylmethoxycarbonylamino)-hexanoyl]-amino}-propionicacid ethyl ester AB (11.5 g, 21.3 mmol) was dissolved in 20% piperidinein dimethylformamide at 0° C. The solution was continued stirring for 1h. The reaction mixture was concentrated under vacuum, water was addedto the residue, and the product was extracted with ethyl acetate. Thecrude product was purified by conversion into its hydrochloride salt.

3-({6-[17-(1,5-Dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yloxycarbonylamino]-hexanoyl}ethoxycarbonylmethyl-amino)-propionicacid ethyl ester AD

The hydrochloride salt of3-[(6-Amino-hexanoyl)-ethoxycarbonylmethyl-amino]-propionic acid ethylester AC (4.7 g, 14.8 mmol) was taken up in dichloromethane. Thesuspension was cooled to 0° C. on ice. To the suspensiondiisopropylethylamine (3.87 g, 5.2 ml, 30 mmol) was added. To theresulting solution cholesteryl chloroformate (6.675 g, 14.8 mmol) wasadded. The reaction mixture was stirred overnight. The reaction mixturewas diluted with dichloromethane and washed with 10% hydrochloric acid.The product was purified by flash chromatography (10.3 g, 92%).

1-{6-[17-(1,5-Dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yloxycarbonylamino]-hexanoyl}-4-oxo-pyrrolidine-3-carboxylicacid ethyl ester AE

Potassium t-butoxide (1.1 g, 9.8 mmol) was slurried in 30 ml of drytoluene. The mixture was cooled to 0° C. on ice and 5 g (6.6 mmol) ofdiester AD was added slowly with stirring within 20 mins. Thetemperature was kept below 5° C. during the addition. The stirring wascontinued for 30 mins at 0° C. and 1 ml of glacial acetic acid wasadded, immediately followed by 4 g of NaH₂PO₄.H₂O in 40 ml of water Theresultant mixture was extracted twice with 100 ml of dichloromethaneeach and the combined organic extracts were washed twice with 10 ml ofphosphate buffer each, dried, and evaporated to dryness. The residue wasdissolved in 60 ml of toluene, cooled to 0° C. and extracted with three50 ml portions of cold pH 9.5 carbonate buffer. The aqueous extractswere adjusted to pH 3 with phosphoric acid, and extracted with five 40ml portions of chloroform which were combined, dried and evaporated todryness. The residue was purified by column chromatography using 25%ethylacetate/hexane to afford 1.9 g of b-ketoester (39%).

[6-(3-Hydroxy-4-hydroxymethyl-pyrrolidin-1-yl)-6-oxo-hexyl]-carbamicacid17-(1,5-dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-ylester AF

Methanol (2 ml) was added dropwise over a period of 1 h to a refluxingmixture of b-ketoester AE (1.5 g, 2.2 mmol) and sodium borohydride(0.226 g, 6 mmol) in tetrahydrofuran (10 ml). Stirring was continued atreflux temperature for 1 h. After cooling to room temperature, 1 N HCl(12.5 ml) was added, the mixture was extracted with ethylacetate (3×40ml). The combined ethylacetate layer was dried over anhydrous sodiumsulfate and concentrated under vacuum to yield the product which waspurified by column chromatography (10% MeOH/CHCl₃) (89%).

(6-{3-[Bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-4-hydroxy-pyrrolidin-1-yl}-6-oxo-hexyl)-carbamicacid17-(1,5-dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-ylester AG

Diol AF (1.25 gm 1.994 mmol) was dried by evaporating with pyridine (2×5ml) in vacuo. Anhydrous pyridine (10 ml) and4,4′-dimethoxytritylchloride (0.724 g, 2.13 mmol) were added withstirring. The reaction was carried out at room temperature overnight.The reaction was quenched by the addition of methanol. The reactionmixture was concentrated under vacuum and to the residue dichloromethane(50 ml) was added. The organic layer was washed with 1M aqueous sodiumbicarbonate. The organic layer was dried over anhydrous sodium sulfate,filtered and concentrated. The residual pyridine was removed byevaporating with toluene. The crude product was purified by columnchromatography (2% MeOH/Chloroform, Rf=0.5 in 5% MeOH/CHCl₃) (1.75 g,95%).

Succinic acidmono-(4-[bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-1-{6-[17-(1,5-dimethyl-hexyl)-10,13-dimethyl2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1Hcyclopenta[a]phenanthren-3-yloxycarbonylamino]-hexanoyl}-pyrrolidin-3-yl)ester AH

Compound AG (1.0 g, 1.05 mmol) was mixed with succinic anhydride (0.150g, 1.5 mmol) and DMAP (0.073 g, 0.6 mmol) and dried in a vacuum at 40°C. overnight. The mixture was dissolved in anhydrous dichloroethane (3ml), triethylamine (0.318 g, 0.440 ml, 3.15 mmol) was added and thesolution was stirred at room temperature under argon atmosphere for 16h. It was then diluted with dichloromethane (40 ml) and washed with icecold aqueous citric acid (5 wt %, 30 ml) and water (2×20 ml). Theorganic phase was dried over anhydrous sodium sulfate and concentratedto dryness. The residue was used as such for the next step.

Cholesterol Derivatised CPG AI

Succinate AH (0.254 g, 0.242 mmol) was dissolved in a mixture ofdichloromethane/acetonitrile (3:2, 3 ml). To that solution DMAP (0.0296g, 0.242 mmol) in acetonitrile (1.25 ml),2,2′-Dithio-bis(5-nitropyridine) (0.075 g, 0.242 mmol) inacetonitrile/dichloroethane (3:1, 1.25 ml) were added successively. Tothe resulting solution triphenylphosphine (0.064 g, 0.242 mmol) inacetonitrile (0.6 ml) was added. The reaction mixture turned brightorange in color. The solution was agitated briefly using a wrist-actionshaker (5 mins). Long chain alkyl amine-CPG (LCAA-CPG) (1.5 g, 61 mM)was added. The suspension was agitated for 2 h. The CPG was filteredthrough a sintered funnel and washed with acetonitrile, dichloromethaneand ether successively. Unreacted amino groups were masked using aceticanhydride/pyridine. The achieved loading of the CPG was measured bytaking UV measurement (37 mM/g).

The synthesis of siRNAs bearing a 5′-12-dodecanoic acid bisdecylamidegroup (herein referred to as “5′-C32-”) or a 5′-cholesteryl derivativegroup (herein referred to as “5′-Chol-”) was performed as described inWO 2004/065601, except that, for the cholesteryl derivative, theoxidation step was performed using the Beaucage reagent in order tointroduce a phosphorothioate linkage at the 5′-end of the nucleic acidoligomer.

Synthesis of dsRNAs conjugated to Chol-p-(GalNAc)₃ (N-acetylgalactosamine-cholesterol) (FIG. 16) and LCO(GalNAc)₃ (N-acetylgalactosamine-3′-Lithocholic-oleoyl) (FIG. 17) is described in U.S.patent application Ser. No. 12/328,528, filed on Dec. 4, 2008, which ishereby incorporated by reference.

Example 3 PCSK9 siRNA Screening in HuH7, HepG2, HeLa and Primary MonkeyHepatocytes Discovers Highly Active Sequences

HuH-7 cells were obtained from JCRB Cell Bank (Japanese Collection ofResearch Bioresources) (Shinjuku, Japan, cat. No.: JCRB0403) Cells werecultured in Dulbecco's MEM (Biochrom AG, Berlin, Germany, cat. No.F0435) supplemented to contain 10% fetal calf serum (FCS) (Biochrom AG,Berlin, Germany, cat. No. S0115), Penicillin 100 U/ml, Streptomycin 100μg/ml (Biochrom AG, Berlin, Germany, cat. No. A2213) and 2 mM L-Glutamin(Biochrom AG, Berlin, Germany, cat. No K0282) at 37° C. in an atmospherewith 5% CO₂ in a humidified incubator (Heraeus HERAcell, KendroLaboratory Products, Langenselbold, Germany). HepG2 and HeLa cells wereobtained from American Type Culture Collection (Rockville, Md., cat. No.HB-8065) and cultured in MEM (Gibco Invitrogen, Karlsruhe, Germany, cat.No. 21090-022) supplemented to contain 10% fetal calf serum (FCS)(Biochrom AG, Berlin, Germany, cat. No. S0115), Penicillin 100 U/ml,Streptomycin 100 μg/ml (Biochrom AG, Berlin, Germany, cat. No. A2213),1× Non Essential Amino Acids (Biochrom AG, Berlin, Germany, cat. No.K-0293), and 1 mM Sodium Pyruvate (Biochrom AG, Berlin, Germany, cat.No. L-0473) at 37° C. in an atmosphere with 5% CO₂ in a humidifiedincubator (Heraeus HERAcell, Kendro Laboratory Products, Langenselbold,Germany).

For transfection with siRNA, HuH7, HepG2, or HeLa cells were seeded at adensity of 2.0×10⁴ cells/well in 96-well plates and transfecteddirectly. Transfection of siRNA (30 nM for single dose screen) wascarried out with lipofectamine 2000 (Invitrogen GmbH, Karlsruhe,Germany, cat. No. 11668-019) as described by the manufacturer.

24 hours after transfection HuH7 and HepG2 cells were lysed and PCSK9mRNA levels were quantified with the Quantigene Explore Kit(Genosprectra, Dumbarton Circle Fremont, USA, cat. No. QG-000-02)according to the protocol. PCSK9 mRNA levels were normalized to GAP-DHmRNA. For each siRNA eight individual datapoints were collected. siRNAduplexes unrelated to PCSK9 gene were used as control. The activity of agiven PCSK9 specific siRNA duplex was expressed as percent PCSK9 mRNAconcentration in treated cells relative to PCSK9 mRNA concentration incells treated with the control siRNA duplex.

Primary cynomolgus monkey hepatocytes (cryopreserved) were obtained fromIn vitro Technologies, Inc. (Baltimore, Md., USA, cat No M00305) andcultured in InVitroGRO CP Medium (cat No Z99029) at 37° C. in anatmosphere with 5% CO₂ in a humidified incubator.

For transfection with siRNA, primary cynomolgus monkey cells were seededon Collagen coated plates (Fisher Scientific, cat. No. 08-774-5) at adensity of 3.5×10⁴ cells/well in 96-well plates and transfecteddirectly. Transfection of siRNA (eight 2-fold dilution series startingfrom 30 nM) in duplicates was carried out with lipofectamine 2000(Invitrogen GmbH, Karlsruhe, Germany, cat. No. 11668-019) as describedby the manufacturer.

16 hours after transfection medium was changed to fresh InVitroGRO CPMedium with Torpedo Antibiotic Mix (In vitro Technologies, Inc, cat. NoZ99000) added.

24 hours after medium change primary cynomolgus monkey cells were lysedand PCSK9 mRNA levels were quantified with the Quantigene Explore Kit(Genosprectra, Dumbarton Circle Fremont, USA, cat. No. QG-000-02)according to the protocol. PCSK9 mRNA levels were normalized to GAPDHmRNA. Normalized PCSK9/GAPDH ratios were then compared to PCSK9/GAPDHratio of lipofectamine 2000 only control.

Tables 1b and 2b (and FIG. 6A) summarize the results and provideexamples of in vitro screens in different cell lines at different doses.Silencing of PCSK9 transcript was expressed as percentage of remainingtranscript at a given dose.

Highly active sequences are those with less than 70% transcriptremaining post treatment with a given siRNA at a dose less than or equalto 100 nM. Very active sequences are those that have less than 60% oftranscript remaining after treatment with a dose less than or equal to100 nM. Active sequences are those that have less than 90% transcriptremaining after treatment with a high dose (100 nM).

Examples of active siRNAs were also screened in vivo in mouse inlipidoid formulations as described below. Active sequences in vitro werealso generally active in vivo (See FIGS. 6A and 6B and example 4).

Example 4 In Vivo Efficacy Screen of PCSK9 siRNAs in Mice

32 PCSK9 siRNAs formulated in LNP-01 liposomes were tested in vivo in amouse model. LNP01 is a lipidoid formulation formed from cholesterol,mPEG2000-C14 Glyceride, and dsRNA. The LNP01 formulation is useful fordelivering dsRNAs to the liver.

Formulation Procedure

The lipidoid LNP-01.4HCl (MW 1487) (FIG. 1), Cholesterol(Sigma-Aldrich), and PEG-Ceramide C16 (Avanti Polar Lipids) were used toprepare lipid-siRNA nanoparticles. Stock solutions of each in ethanolwere prepared: LNP-01, 133 mg/ml; Cholesterol, 25 mg/ml, PEG-CeramideC16, 100 mg/ml. LNP-01, Cholesterol, and PEG-Ceramide C16 stocksolutions were then combined in a 42:48:10 molar ratio. Combined lipidsolution was mixed rapidly with aqueous siRNA (in sodium acetate pH 5)such that the final ethanol concentration was 35-45% and the finalsodium acetate concentration was 100-300 mM. Lipid-siRNA nanoparticlesformed spontaneously upon mixing. Depending on the desired particle sizedistribution, the resultant nanoparticle mixture was in some casesextruded through a polycarbonate membrane (100 nm cut-off) using athermobarrel extruder (Lipex Extruder, Northern Lipids, Inc). In othercases, the extrusion step was omitted. Ethanol removal and simultaneousbuffer exchange was accomplished by either dialysis or tangential flowfiltration. Buffer was exchanged to phosphate buffered saline (PBS) pH7.2.

Characterization of Formulations

Formulations prepared by either the standard or extrusion-free methodare characterized in a similar manner. Formulations are firstcharacterized by visual inspection. They should be whitish translucentsolutions free from aggregates or sediment. Particle size and particlesize distribution of lipid-nanoparticles are measured by dynamic lightscattering using a Malvern Zetasizer Nano ZS (Malvern, USA). Particlesshould be 20-300 nm, and ideally, 40-100 nm in size. The particle sizedistribution should be unimodal. The total siRNA concentration in theformulation, as well as the entrapped fraction, is estimated using a dyeexclusion assay. A sample of the formulated siRNA is incubated with theRNA-binding dye Ribogreen (Molecular Probes) in the presence or absenceof a formulation disrupting surfactant, 0.5% Triton-X100. The totalsiRNA in the formulation is determined by the signal from the samplecontaining the surfactant, relative to a standard curve. The entrappedfraction is determined by subtracting the “free” siRNA content (asmeasured by the signal in the absence of surfactant) from the totalsiRNA content. Percent entrapped siRNA is typically >85%.

Bolus Dosing

Bolus dosing of formulated siRNAs in C57/BL6 mice (5/group, 8-10 weeksold, Charles River Laboratories, MA) was performed by tail veininjection using a 27 G needle. SiRNAs were formulated in LNP-01 (andthen dialyzed against PBS) at 0.5 mg/ml concentration allowing thedelivery of the 5 mg/kg dose in 10 μl/g body weight. Mice were keptunder an infrared lamp for approximately 3 min prior to dosing to easeinjection.

48 hour post dosing mice were sacrificed by CO₂-asphyxiation. 0.2 mlblood was collected by retro-orbital bleeding and the liver washarvested and frozen in liquid nitrogen. Serum and livers were stored at−80° C. μl

Frozen livers were grinded using 6850 Freezer/Mill Cryogenic Grinder(SPEX CentriPrep, Inc) and powders stored at −80° C. until analysis.

PCSK9 mRNA levels were detected using the branched-DNA technology basedkit from QuantiGene Reagent System (Genospectra) according to theprotocol. 10-20 mg of frozen liver powders was lysed in 600 μl of 0.16μg/ml Proteinase K (Epicentre, #MPRK092) in Tissue and Cell LysisSolution (Epicentre, #MTC096H) at 65° C. for 3 hours. Then 10 μl of thelysates were added to 90 μl of Lysis Working Reagent (1 volume of stockLysis Mixture in two volumes of water) and incubated at 52° C. overnighton Genospectra capture plates with probe sets specific to mouse PCSK9and mouse GAPDH or cyclophilin B. Nucleic acid sequences for CaptureExtender (CE), Label Extender (LE) and blocking (BL) probes wereselected from the nucleic acid sequences of PCSK9, GAPDH and cyclophilinB with the help of the QuantiGene ProbeDesigner Software 2.0(Genospectra, Fremont, Calif., USA, cat. No. QG-002-02). Chemoluminescence was read on a Victor2-Light (Perkin Elmer) as Relativelight units. The ratio of PCSK9 mRNA to GAPDH or cyclophilin B mRNA inliver lysates was averaged over each treatment group and compared to acontrol group treated with PBS or a control group treated with anunrelated siRNA (blood coagulation factor VII).

Total serum cholesterol in mouse serum was measured using the StanBioCholesterol LiquiColor kit (StanBio Laboratory, Boerne, Tex., USA)according to manufacturer's instructions. Measurements were taken on aVictor2 1420 Multilabel Counter (Perkin Elmer) at 495 nm.

Results

At least 10 PCSK9 siRNAs showed more than 40% PCSK9 mRNA knock downcompared to a control group treated with PBS, while control grouptreated with an unrelated siRNA (blood coagulation factor VII) had noeffect (FIGS. 2-3). Silencing of PCSK9 transcript also correlated with alowering of total serum cholesterol in these animals (FIGS. 4-5). Themost efficacious siRNAs with respect to knocking down PCSK9 mRNAs alsoshowed the most pronounced cholesterol lowering effects (compare FIGS.2-3 and FIGS. 4-5). In addition there was a strong correlation betweenthose molecules that were active in vitro and those active in vivo(compare FIGS. 6A and 6B).

Sequences containing different chemical modifications were also screenedin vitro (Tables 1 and 2) and in vivo. As an example, less modifiedsequences AD-9314 and AD-9318, and a more modified versions of thatsequence AD-9314 (AD-10792, AD-10793, and AD-10796); AD-9318-(AD-10794,AD-10795, AD-10797) were tested both in vitro (in primary monkeyhepatocytes) or in vivo (AD-9314 and AD-10792) formulated in LNP-01.FIG. 7 (also see Tables 1 and 2) shows that the parent molecules AD-9314and AD-9318 and the modified versions were all active in vitro. FIG. 8as an example shows that both the parent AD-9314 and the more highlymodified AD-10792 sequences were active in vivo displaying 50-60%silencing of endogenous PCSK9 in mice. FIG. 9 further exemplifies thatactivity of other chemically modified versions of AD-9314 and AD-0792.

AD-3511, a derivative of AD-10792, was as efficacious as 10792 (IC50 of˜0.07-0.2 nM) (data not shown). The sequences of the sense and antisensestrands of AD-3511 are as follows:

Sense strand: SEQ ID NO: 1521 5′- GccuGGAGuuuAuucGGAAdTsdTAntisense strand: SEQ ID NO: 1522 5′- puUCCGAAuAAACUCcAGGCdTsdT

Example 5 PCSK9 Duration of Action Experiments in Rats and NHP

Rats

Rats were treated via tail vein injection with 5 mg/kg of LNP01-10792(Formulated ALDP-10792). Blood was drawn at the indicated time points(see Table 3) and the amount of total cholesterol compared to PBStreated animals was measured by standard means. Total cholesterol levelsdecreased at day two ˜60% and returned to baseline by day 28. These datashow that formulated versions of PCSK9 siRNAs lower cholesterol levelsfor extended periods of time.

Monkeys

Cynomolgus monkeys were treated with LNP01 formulated dsRNA and LDL-Clevels were evaluated. A total of 19 cynomolgus monkeys were assigned todose groups. Beginning on Day −11, animals were limit-fed twice-a-dayaccording to the following schedule: feeding at 9 a.m., feed removal at10 a.m., feeding at 4 p.m., feed removal at 5 p.m. On the first day ofdosing all animals were dosed once via 30-minute intravenous infusion.The animals were evaluated for changes in clinical signs, body weight,and clinical pathology indices, including direct LDL and HDLcholesterol.

Venipuncture through the femoral vein was used to collect blood samples.Samples were collected prior to the morning feeding (i.e., before 9a.m.) and at approximately 4 hours (beginning at 1 p.m.) after themorning feeding on Days −3, −1, 3, 4, 5, and 7 for Groups 1-7; on Day 14for Groups 1, 4, and 6; on Days 18 and 21 for Group 1; and on Day 21 forGroups 4 and 6. At least two 1.0 ml samples were collected at each timepoint.

No anticoagulant was added to the 1.0 ml serum samples, and the dryanticoagulant Ethylenediaminetetraacetic acid (K2) was added to each 1.0ml plasma sample. Serum samples were allowed to stand at roomtemperature for at least 20 minutes to facilitate coagulation and thenthe samples were placed on ice. Plasma samples were placed on ice assoon as possible following sample collection. Samples were transportedto the clinical pathology lab within 30 minutes for further processing.

Blood samples were processed to serum or plasma as soon as possibleusing a refrigerated centrifuge, per Testing Facility Standard operatingprocedure. Each sample was split into 3 approximately equal volumes,quickly frozen in liquid nitrogen, and placed at −70° C. Each aliquotshould have had a minimum of approximately 50 μL. If the total samplevolume collected was under 150 μL, the residual sample volume went intothe last tube. Each sample was labeled with the animal number, dosegroup, day of collection, date, nominal collection time, and studynumber(s). Serum LDL cholesterol was measured directly per standardprocedures on a Beckman analyzer according to manufactures instructions.

The results are shown in Table 4. LNP01-10792 and LNP01-9680administered at 5 mg/kg decreased serum LDL cholesterol within 3 to 7days following dose administration. Serum LDL cholesterol returned tobaseline levels by Day 14 in most animals receiving LNP01-10792 and byDay 21 in animals receiving LNP01-9680. This data demonstrated a greaterthan 21 day duration of action for cholesterol lowering of LNP01formulated ALDP-9680.

Example 6 PCSK9 siRNAs Cause Decreased PCSK mRNA in Liver Extracts, andLower Serum Cholesterol Levels in Mice and Rats

To test if acute silencing of the PCSK9 transcript by a PCSK9 siRNA (andsubsequent PCSK9 protein down-regulation), would result in acutely lowertotal cholesterol levels, siRNA molecule AD-1a2 (AD-10792) wasformulated in an LNP01 lipidoid formulation. Sequences and modificationsof these dsRNAs are shown in Table 5a. Liposomal formulated siRNA duplexAD-1a2 (LNP01-1a2) was injected via tail vein in low volumes (˜0.2 mlfor mouse and ˜1.0 ml for rats) at different doses into C57/BL6 mice orSprague Dawley rats.

In mice, livers were harvested 48 hours post-injection, and levels ofPCSK9 transcript were determined. In addition to liver, blood washarvested and subjected to a total cholesterol analysis. LNP01-1a2displayed a clear dose response with maximal PCSK9 message suppression(˜60-70%) as compared to a control siRNA targeting luciferase(LNP01-ctrl) or PBS treated animals (FIG. 14A). The decrease of PCSK9transcript at the highest dose translated into a ˜30% lowering of totalcholesterol in mice (FIG. 14B). This level of cholesterol reduction isbetween that reported for heterozygous and homozygous PCSK9 knock-outmice (Rashid et al., Proc. Natl. Acad. Sci. USA 102:5374-9, 2005, epubApr. 1, 2005). Thus, lowering of PCSK9 transcript through an RNAimechanism is capable of acutely decreasing total cholesterol in mice.Moreover the effect on the PCSK9 transcript persisted between 20-30days, with higher doses displaying greater initial transcript levelreduction, and subsequently more persistent effects.

Down-modulation of total cholesterol in rats has been historicallydifficult as cholesterol levels remain unchanged even at high doses ofHMG-CoA reductase inhibitors. Interestingly, as compared to mice, ratsappear to have a much higher level of PCSK9 basal transcript levels asmeasured by bDNA assays. Rats were dosed with a single injection ofLNP01-a2 via tail vein at 1, 2.5 and 5 mg/kg. Liver tissue and bloodwere harvested 72 hours post-injection. LNP01-1a2 exhibited a clear doseresponse effect with maximal 50-60% silencing of the PCSK9 transcript atthe highest dose, as compared to a control luciferase siRNA and PBS(FIG. 10A). The mRNA silencing was associate with an acute ˜50-60%decrease of serum total cholesterol (FIGS. 10A and 10B) lasting 10 days,with a gradual return to pre-dose levels by ˜3 weeks (FIG. 10B). Thisresult demonstrated that lowering of PCSK9 via siRNA targeting hadacute, potent and lasting effects on total cholesterol in the rat modelsystem. To confirm that the transcript reduction observed was due to asiRNA mechanism, liver extracts from treated or control animals weresubjected to 5′ RACE, a method previously utilized to demonstrate thatthe predicted siRNA cleavage event occurs (Zimmermann et al., Nature.441:111-4, 2006, Epub 2006 Mar. 26). PCR amplification and detection ofthe predicted site specific mRNA cleavage event was observed in animalstreated with LNP01-1a2, but not PBS or LNP01-ctrl control animals.(Frank-Kamanetsky et al. (2008) PNAS 105:119715-11920) This resultdemonstrated that the effects of LNP01-1a2 observed were due to cleavageof the PCSK9 transcript via an siRNA specific mechanism.

The mechanism by which PCSK9 impacts cholesterol levels has been linkedto the number of LDLRs on the cell surface. Rats (as opposed to mice,NHP, and humans) control their cholesterol levels through tightregulation of cholesterol synthesis and to a lesser degree through thecontrol of LDLR levels. To investigate whether modulation of LDLR wasoccurring upon RNAi therapeutic targeting of PCSK9, we quantified theliver LDLR levels (via western blotting) in rats treated with 5 mg/kgLNP01-1a2. As shown in FIG. 11, LNP01-1a2 treated animals had asignificant (˜3-5 fold average) induction of LDLR levels 48 hours post asingle dose of LNP01-1a2 compared to PBS or LNP01-ctrl control siRNAtreated animals.

Assays were also performed to test whether reduction of PCSK9 changesthe levels of triglycerides and cholesterol in the liver itself. Acutelowering of genes involved in VLDL assembly and secretion such asmicrosomal triglyceride transfer protein (MTP) or ApoB by geneticdeletion, compounds, or siRNA inhibitors results in increased livertriglycerides (see, e.g., Akdim et al., Curr. Opin. Lipidol. 18:397-400,2007). Increased clearance of plasma cholesterol induced by PCSK9silencing in the liver (and a subsequent increase in liver LDLR levels)was not predicted to result in accumulation of liver triglycerides.However, to address this possibility, liver cholesterol and triglycerideconcentrations in livers of the treated or control animals werequantified. As shown in FIG. 10C, there was no statistical difference inliver TG levels or cholesterol levels of rats administered PCSK9 siRNAscompared to the controls. These results indicated that PCSK9 silencingand subsequent cholesterol lowering is unlikely to result in excesshepatic lipid accumulation.

Example 7 Additional Modifications to siRNAs do not Affect Silencing andDuration of Cholesterol Reduction in Rats

Phosphorothioate modifications at the 3′ ends of both sense andantisense strands of a dsRNA can protect against exonucleases. 2′OMe and2′F modifications in both the sense and antisense strands of a dsRNA canprotect against endonucleases. AD-1a2 (see Table 5b) contains 2′OMemodifications on both the sense and antisense strands. Experiments wereperformed to determine if the inherent stability (as measured by siRNAstability in human serum) or the degree or type of chemical modification(2′OMe versus 2′F or a mixture) was related to either the observed ratefficacy or the duration of silencing effects. Stability of siRNAs withthe same AD-1a2 core sequence, but containing different chemicalmodifications were created and tested for activity in vitro in primaryCyno monkey hepatocytes. A series of these molecules that maintainedsimilar activity as measured by in vitro IC50 values for PCSK9 silencing(Table 5b), were then tested for their stability against exo andendonuclease cleavage in human serum. Each duplex was incubated in humanserum at 37° C. (a time course), and subjected to HPLC analysis. Theparent sequence AD-1a2 had a T1/2 of ˜7 hours in pooled human serum.Sequences AD-1a3, AD-1a5, and AD-1a4, which were more heavily modified(see chemical modifications in Table 5) all had T 1/2's greater than 24hours. To test whether the differences in chemical modification orstability resulted in changes in efficacy, AD-1a2, AD-1a3, AD-1a5,AD-1a4, and an AD-control sequence were formulated and injected intorats. Blood was collected from animals at various days post-dose, andtotal cholesterol concentrations were measured. Previous experiments hadshown a very tight correlation between the lowering of PCSK9 transcriptlevels and total cholesterol values in rats treated with LNP01-1a2 (FIG.10A). All four molecules were observed to decrease total cholesterol by˜60% day 2 post-dose (versus PBS or control siRNA), and all of themolecules had equal effects on total cholesterol levels displayingsimilar magnitude and duration profiles. There was no statisticaldifference in the magnitude of cholesterol lowering and the duration ofeffect demonstrated by these molecules, regardless of their differentchemistries or stabilities in human serum.

Example 8 LNP01-1a2 and LNP01-3a1 Silence Human PCSK9 and CirculatingHuman PCSK9 Protein in Transgenic Mice

The efficacy of LNP01-1a2 (i.e., PCS-A2 or AD-10792) and anothermolecule, AD-3a1 (i.e., PCS-C2 or AD-9736) (which targets only human andmonkey PCSK9 message), to silence the human PCSK9 gene was tested invivo. A line of transgenic mice expressing human PCSK9 under the ApoEpromoter was used (Lagace et al., J Clin Invest. 116:2995-3005, 2006).Specific PCR reagents and antibodies were designed that detected thehuman but not the mouse transcripts and protein respectively. Cohorts ofthe humanized mice were injected with a single dose of LNP01-1a2 (a.k.a.LNP-PCS-A2) or LNP01-3a1 (a.k.a. LNP-PCS-C2), and 48 hours later bothlivers and blood were collected. A single dose of LNP01-1a2 or LNP01-3a1was able to decrease the human PCSK9 transcript levels by >70% (FIG.15A), and this transcript down-regulation resulted in significantlylower levels of circulating human PCSK9 protein as measured by ELISA(FIG. 15B). These results demonstrated that both siRNAs were capable ofsilencing the human transcript and subsequently reducing the amount ofcirculating plasma human PCSK9 protein.

Example 9 Secreted PCSK9 Levels are Regulated by Diet in NHP

In mice, PCSK9 mRNA levels are regulated by the transcription factorsterol regulatory element binding protein-2 and are reduced by fasting.In clinical practice, and standard NHP studies, blood collection andcholesterol levels are measured after an overnight fasting period. Thisis due in part to the potential for changes in circulating TGs tointerfere with the calculation of LDLc values. Given the regulation ofPCSK9 levels by fasting and feeding behavior in mice, experiments wereperformed to understand the effect of fasting and feeding in NHP.

Cyno monkeys were acclimated to a twice daily feeding schedule duringwhich food was removed after a one hour period. Animals were fed from9-10 am in the morning, after which food was removed. The animals werenext fed once again for an hour between 5 pm-6 pm with subsequent foodremoval. Blood was drawn after an overnight fast (6 pm until 9 am thenext morning), and again, 2 and 4 hours following the 9 am feeding.PCSK9 levels in blood plasma or serum were determined by ELISA assay(see Methods). Interestingly, circulating PCSK9 levels were found to behigher after the overnight fasting and decreased 2 and 4 hours afterfeeding. This data was consistent with rodent models where PCSK9 levelswere highly regulated by food intake. However, unexpectedly, the levelsof PCSK9 went down the first few hours post-feeding. This result enableda more carefully designed NHP experiment to probe the efficacy offormulated AD-1a2 and another PCSK9 siRNA (AD-2a1) that was highlyactive in primary Cyno hepatocytes.

Example 10 PCSK9 siRNAs Reduce Circulating LDLc, ApoB, and PCSK9, butnot HDLc in Non-Human Primates (NHPs)

siRNAs targeting PCSK9 acutely lowered both PCSK9 and total cholesterollevels by 72 hours post-dose and lasted ˜21-30 days after a single dosein mice and rats. To extend these findings to a species whoselipoprotein profiles most closely mimic that of humans, furtherexperiments were performed in the Cynomologous (Cyno) monkey model.

siRNA 1 (LNP01-10792) and siRNA 2 (LNP-01-9680), both targeting PCSK9were administered to cynomologous monkeys. As shown in FIG. 12, bothsiRNAs caused significant lipid lowering for up to 7 days postadministration. siRNA 2 caused ˜50% lipid lowering for at least 7 dayspost-administration, and ˜60% lipid lowering at day 14post-administration, and siRNA 1 caused ˜60% LDLc lowering for at least7 days.

Male Cynos were first pre-screened for those that had LDLc of 40 mg/dlor higher. Chosen animals were then put on a fasted/fed diet regime andacclimated for 11 days. At day −3 and −1 pre-dose, serum was drawn atboth fasted and 4 hours post-fed time points and analyzed for totalcholesterol (Tc), LDL (LDLc), HDL cholesterol (HDLc) as well astriglycerides (TG), and PCSK9 plasma levels. Animals were randomizedbased on their day −3 LDLc levels. On the day of dosing (designated day1), either 1 mg/kg or 5 mg/kg of LNP01-1a2 and 5 mg/kg LNP01-2a1 wereinjected, along with PBS and 1 mg/kg LNP01-ctrl as controls. All doseswere well tolerated with no in-life findings. As the experimentprogressed it became apparent (based on LDLc lowering) that the lowerdose was not efficacious. We therefore dosed the PBS group animals onday 14 with 5 mg/kg LNP01-ctrl control siRNA, which could then serve asan additional control for the high dose groups of 5 mg/kg LNP01-1a2 and5 mg/kg LNP01-2a1. Initially blood was drawn from animals on days 3, 4,5, and 7 post-dose and Tc, HDLc, LDLc, and TGs concentrations weremeasured. Additional blood draws from the LNP01-1a2, LNP01-2a1 high dosegroups were carried out at day 14 and day 21 post-dose (as the LDLclevels had not returned to baseline by day 7).

As shown in FIG. 12A, a single dose of LNP01-1a2 or LNP01-2a1 resultedin a statistically significant reduction of LDLc beginning at day 3post-dose that returned to baseline over ˜14 days (for LNP01-1a2) and˜21 days (LNP01-2a1). This effect was not seen in either the PBS, thecontrol siRNA groups, or the 1 mg/kg treatment groups. LNP01-2a1resulted in an average lowering of LDLc of 56% 72 hours post-dose, with1 of 4 animals achieving nearly 70% LDLc, and all others achieving >50%LDLc decrease, as compared to pre-dose levels, (see FIG. 12A. Asexpected, the lowering of LDLc in the treated animals also correlatedwith a reduction of circulating ApoB levels as measured by serum ELISA(FIG. 12B). Interestingly, the degree of LDLc lowering observed in thisstudy of Cyno monkey was greater than those that have been reported forhigh dose statins, as well as, for other current standard of carecompounds used for hypercholesterolemia. The onset of action is alsomuch more acute than that of statins with effects being seen as early as48 hours post-dose.

Neither LNP01-1a2 nor LNP01-2a1 treatments resulted in a lowering ofHDLc. In fact, both molecules resulted (on average) in a trend towards adecreased Tc/HDL ratio (FIG. 12C). In addition, circulating triglyceridelevels, and with the exception of one animal, ALT and AST levels werenot significantly impacted.

PCSK9 protein levels were also measured in treated and control animals.As shown in FIG. 11, LNP01-1a2 and LNP01-2a1 treatment each resulted intrends toward decreased circulating PCSK9 protein levels versuspre-dose. Specifically, the more active siRNA LNP01-2a1 demonstratedsignificant reduction of circulating PCSK9 protein versus both PBS (day3-21) and LNP01-ctrl siRNA control (day 4, day 7).

Example 11 Modified siRNA and Activation of Immune Responses in hPBMCs

siRNAs were tested for activation of the immune system in primary humanblood monocytes (hPBMC). Two control inducing sequences and theunmodified parental compound AD-1a1 was found to induce both IFN-alphaand TNF-alpha. However, chemically modified versions of this sequence(AD-1a2, AD-1a3, AD-1a5, and AD-1a4) as well as AD-2a1 were negative forboth IFN-alpha and TNF-alpha induction in these same assays (see Table5, and FIGS. 13A and 13B). Thus chemical modifications are capable ofdampening both IFN-alpha and TNF-alpha responses to siRNA molecules. Inaddition, neither AD-1a2, nor AD-2a1 activated IFN-alpha when formulatedinto liposomes and tested in mice.

Example 12 Evaluation of siRNA Conjugates in Mice

AD-10792 was conjugated to GalNAc)3/Cholesterol (FIG. 16) orGalNAc)3/LCO (FIG. 17). The sense strand was synthesized with theconjugate on the 3′ end. The conjugated siRNAs were assayed for effectson PCSK9 transcript levels and total serum cholesterol in mice using themethods described below.

Briefly, mice were dosed via tail injection with one of the 2 conjugatedsiRNAs or PBS on three consecutive days: day 0, day 1 and day 2 with adosage of about 100, 50, 25 or 12.5 mg/kg. Each dosage group included 6mice. 24 hour post last dosing mice were sacrificed and blood and liversamples were obtained, stored, and processed to determine PCSK9 mRNAlevels and total serum cholesterol.

The results are shown in FIG. 18. Compared to control PBS, both siRNAconjugates demonstrated activity with an ED50 of 3×50 mg/kg forGalNAc)3/Cholesterol conjugated AD-10792 and 3×100 mg/kg forGalNAc)3/LCO conjugated AD-10792. The results indicate that Cholesterolconjugated siRNA with GalNAc are active and capable of silencing PCSK9in the liver resulting in cholesterol lowering.

Bolus Dosing

Bolus dosing of formulated siRNAs in C57/BL6 mice (6/group, 8-10 weeksold, Charles River Laboratories, MA) was performed by tail veininjection using a 27 G needle. SiRNAs were formulated in LNP-01 (andthen dialyzed against PBS) and diluted with PBS to concentrations 1.0,0.5, 0.25 and 0.125 mg/ml allowing the delivery of 100; 50; 25 and 12.5mg/kg doses in 10 μl/g body weight. Mice were kept under an infraredlamp for approximately 3 min prior to dosing to ease injection.

24 hour post last dose mice were sacrificed by CO2-asphyxiation. 0.2 mlblood was collected by retro-orbital bleeding and the liver washarvested and frozen in liquid nitrogen. Serum and livers were stored at−80° C. Frozen livers were grinded using 6850 Freezer/Mill CryogenicGrinder (SPEX CentriPrep, Inc) and powders stored at −80° C. untilanalysis.

PCSK9 mRNA levels were detected using the branched-DNA technology basedkit from QuantiGene Reagent System (Panomics, USA) according to theprotocol. 10-20 mg of frozen liver powders was lysed in 600 μl of 0.16μg/ml Proteinase K (Epicentre, #MPRK092) in Tissue and Cell LysisSolution (Epicentre, #MTC096H) at 65° C. for 3 hours. Then 10 μl of thelysates were added to 90 μl of Lysis Working Reagent (1 volume of stockLysis Mixture in two volumes of water) and incubated at 52° C. overnighton Genospectra capture plates with probe sets specific to mouse PCSK9and mouse GAPDH. Probes sets for mouse PCSK9 and mouse GAPDH werepurchased from Panomics, USA. Chemo luminescence was read on aVictor2-Light (Perkin Elmer) as Relative light units. The ratio of PCSK9mRNA to mGAPDH mRNA in liver lysates was averaged over each treatmentgroup and compared to a control group treated with PBS or a controlgroup treated with an unrelated siRNA (blood coagulation factor VII).

Total serum cholesterol in mouse serum was measured using the TotalCholesterol Assay (Wako, USA) according to manufacturer's instructions.Measurements were taken on a Victor2 1420 Multilabel Counter (PerkinElmer) at 600 nm.

Example 13 Evaluation of Lipid Formulated siRNAs in Rats

Briefly, rats were dosed via tail injection with SNALP formulated siRNAsor PBS with a single dosage of about 0.3, 1.0, and 3.0 mg/kg of SNALPformulated AD-10792. Each dosage group included 5 rats. 72 hour postdosing rats were sacrificed and blood and liver samples were obtained,stored, and processed to determine PCSK9 mRNA and total serumcholesterol levels. The results are shown in FIG. 19. Compared tocontrol PBS, SNALP formulated AD-10792 (FIG. 19A) had an ED50 of about1.0 mg/kg for both lowering of PCSK9 transcript levels and total serumcholesterol levels. These results show that administration of SNALPformulated siRNA results in effective and efficient silencing of PCSK9and subsequent lowering of total cholesterol in vivo.

Bolus Dosing

Bolus dosing of formulated siRNAs in Sprague-Dawley rats (5/group,170-190 g body weight, Charles River Laboratories, MA) was performed bytail vein injection using a 27 G needle. SiRNAs were formulated in SNALP(and then dialyzed against PBS) and diluted with PBS to concentrations0.066; 0.2 and 0.6 mg/ml allowing the delivery of 0.3; 1.0 and 3.0 mg/kgof SNALP formulated AD-10792 in 5 μl/g body weight. Rats were kept underan infrared lamp for approximately 3 min prior to dosing to easeinjection.

72 hour post last dose rats were sacrificed by CO2-asphyxiation. 0.2 mlblood was collected by retro-orbital bleeding and the liver washarvested and frozen in liquid nitrogen. Serum and livers were stored at−80° C. Frozen livers were grinded using 6850 Freezer/Mill CryogenicGrinder (SPEX CentriPrep, Inc) and powders stored at −80° C. untilanalysis.

PCSK9 mRNA levels were detected using the branched-DNA technology basedkit from QuantiGene Reagent System (Panomics, USA) according to theprotocol. 10-20 mg of frozen liver powders was lysed in 600 μl of 0.16μg/ml Proteinase K (Epicentre, #MPRK092) in Tissue and Cell LysisSolution (Epicentre, #MTC096H) at 65° C. for 3 hours. Then 10 μl of thelysates were added to 90 μl of Lysis Working Reagent (1 volume of stockLysis Mixture in two volumes of water) and incubated at 52° C. overnighton Genospectra capture plates with probe sets specific to rat PCSK9 andrat GAPDH. Probes sets for rat PCSK9 and rat GAPDH were purchased fromPanomics, USA. Chemo luminescence was read on a Victor2-Light (PerkinElmer) as Relative light units. The ratio of rat PCSK9 mRNA to rat GAPDHmRNA in liver lysates was averaged over each treatment group andcompared to a control group treated with PBS or a control group treatedwith an unrelated siRNA (blood coagulation factor VII).

Total serum cholesterol in rat serum was measured using the TotalCholesterol Assay (Wako, USA) according to manufacturer's instructions.Measurements were taken on a Victor2 1420 Multilabel Counter (PerkinElmer) at 600 nm.

Example 14 In Vitro Efficacy Screen in HeLa Cells of Mismatch Walk ofAD-9680 and AD-14676

The effects of variations in sequence or modification on theeffectiveness of AD-9680, AD-14676, and AD-10792 were assayed in HeLacells. A number of variants were synthesized as shown in Table 6 andinclude adding DFT (2,4-Difluorotoluoyl, a thymidine triphosphate shapeanalog lacking Watson-Crick pairing); adding single or combinationmismatches; and testing two different backbone chemistries: leading witha 2′-O methyl, or alternating with 2′F.

Sequences of the 3 parent duplexes can be found in Table 1a and areduplicated as follows:

SEQ SEQ target ID ID region Sense strand (5′ to 3′) NO:Antisense strand (5′ to 3′) NO: Duplex 3530- uucuAGAccuGuuuuGcuuTsT 1229AAGcAAAAcAGGUCuAGAATsT 1230 AD- 3548 9680 3530- UfuCfuAfgAfcCfuGfuUfuUfg1231 p- 1232 AD- 3548 CfuUfTsT aAfgCfaAfaAfcAfgGfuCfuAfgA 14676 faTsT1091- GccuGGAGuuuAuucGGAATsT 459 UUCCGAAuAAACUCcAGGCTsT 460 AD- 110910792

HeLa were plated in 96-well plates (8,000-10,000 cells/well) in 100 μl10% fetal bovine serum in Dulbecco's Modified Eagle Medium (DMEM). Whenthe cells reached approximately 50% confluence (˜24 hours later) theywere transfected with serial four-fold dilutions of siRNA starting at 10nM. 0.4 μl of transfection reagent Lipofectamine™ 2000 (InvitrogenCorporation, Carlsbad, Calif.) was used per well and transfections wereperformed according to the manufacturer's protocol. Namely, the siRNA:Lipofectamine™ 2000 complexes were prepared as follows. The appropriateamount of siRNA was diluted in Opti-MEM I Reduced Serum Medium withoutserum and mixed gently. The Lipofectamine™ 2000 was mixed gently beforeuse, then for each well of a 96 well plate 0.4 μl was diluted in 25 μlof Opti-MEM I Reduced Serum Medium without serum and mixed gently andincubated for 5 minutes at room temperature. After the 5 minuteincubation, 1 μl of the diluted siRNA was combined with the dilutedLipofectamine™ 2000 (total volume is 26.4 μl). The complex was mixedgently and incubated for 20 minutes at room temperature to allow thesiRNA: Lipofectamine™ 2000 complexes to form. Then 100 μl of 10% fetalbovine serum in DMEM was added to each of the siRNA:Lipofectamine™ 2000complexes and mixed gently by rocking the plate back and forth. 100 μlof the above mixture was added to each well containing the cells and theplates were incubated at 37° C. in a CO2 incubator for 24 hours, thenthe culture medium was removed and 100 μA 10% fetal bovine serum in DMEMwas added.

24 hours post medium change medium was removed, cells were lysed andcell lysates assayed for PCSK9 mRNA silencing by bDNA assay (Panomics,USA) following the manufacturer's protocol. Chemo luminescence was readon a Victor2-Light (Perkin Elmer) as Relative light units. The ratio ofhuman PCSK9 mRNA to human GAPDH mRNA in cell lysates was compared tothat of cells treated with Lipofectamine™ 2000 only control.

FIG. 20 is dose response curves of a series of compounds related toAD-9680. FIG. 21 is a dose response curve of a series of compoundsrelated to AD-14676 The results show that DFTs or mismatches in certainpositions are able increase the activity (as evidenced by lower IC50values) of both parent compounds. FIG. 24 is a dose response curvecomparing the efficiency of parent duplexes AD-9680 and AD-10792 withmodified duplexes wherein a DFT is inserted at position 10 of the sensestrand. This modification improves the efficiency by about 2 fold inHeLa cells.

Without being bound by theory, it is hypothesized that destabilizationof the sense strand through the introduction of mismatches, or DFT mightresult in quicker removal of the sense strand.

Example 15 Lack of Off Target Effects in Hep3B Cells at HighConcentrations

A lipid formulated PCSK9 targeted siRNA (AD-9680) was transfected intoHep3B cells at concentrations of 250 nM, 1 uM and 5 uM in triplicatesusing the reagent RNAiMAX (Invitrogen) according to the manufacture'sinstruction: 1 ul of transfection reagent; reverse transfectionprotocol. Samples were collected 48 hrs post transfection. Total RNA waspurified using MagMAX™-96 Total RNA Isolation Kit (Ambion); cDNA wassynthesized with High Capacity cDNA Reverse Transcription Kit with RNaseInhibitor (ABI) from 13.5 μl of RNA prep; ABI Gene Expression Taqmanassays were used; q-PCR reactions were set up according tomanufacturer's instruction using TaqMan® Gene Expression Master Mix(ABI) and run on ABI 7900 machine. Delta delta Ct method was used tocalculate values. Samples were normalized to hGAPDH and calibrated tomock transfection.

Transcript levels were measured for the following genes having theclosest homology to the target sequence: ORMDL2, BMP6, TAPT1, MYEF2,LOC442252, RFT1, and PCSK9.

The results are shown in FIG. 22. No off target effects were observed athigh concentrations of dsRNA (PCS-B2=AD-9680).

AD-9680 S 1531 uucuAGAccuGuuuuGcuudTsdT AS 1532 AAGcAAAAcAGGUCuAGAAdTsdT

Example 16 Maintenance of Decrease in Total Cholesterol Levels in Ratsby Lower Dosage of AD-10792

Rats were treated with 3 mg/kg bolus dose of SNALP-Dlin DMA formulatedAD-10792. At day 2, total serum cholesterol levels were determined. Thiswas followed by once a week dosing at 1.0 and 0.3 mg/kg for four weeks.Rats were bled one day prior to repeat dosing and total serumcholesterol levels were determined. The negative control was PBS.

The results are shown in the graph of FIG. 23. After 3 mg/kg bolus dose,total cholesterol levels decreased by 60% and were maintained at about50% by repeated once a week 1.0 and 0.3 mg/kg dosing and come back topre dose levels after repeated dosing is stopped.

A 10 fold lower (than EC50), once a week, maintenance dose effectivelymaintains silencing with cholesterol levels returning to baseline by 15days post last injection. The initial does of PCSK9 increased LDLRlevels as reflected by the decrease in total serum cholesterol. Thisincrease in LDLR levels increased the efficacy of the PCSK9 targetedsiRNA as reflected by the lower dosage of subsequent administrationAD-10792.

Example 17 Assay of Effects on Cholesterol Levels in Rats afterAdministration of Various Lipid Formulations of AD-10792

Rats were treated with four different lipid formulations of AD-10792including SNALP and LNP08, described herein. At day 3, total serumcholesterol levels were determined. The experiment was performed usingthe methods described herein. Administration of LNP-08 formulatedAD-10792 results in the lowest EC50 of 0.08 mg/kg compared to LNP01formulated (EC50 of 2.0 mg/kg) and SNALP formulated (EC50 of 1.0 mg/kg).(data not shown).

Example 18 PCSK9 siRNA Tiling Experiment

Bioinformatic Selection of PCSK9 Tiling Set

Sense and antisense oligomers were designed to target the human PCSK9transcript in the flanking regions immediately upstream and downstreamof the 19 base target region of ALN-PCSK9 (AD-9680). We used the NCBIRefseq NM_(—)174936.2 as the reference human transcript for the PCSK9gene. The antisense oligo of AD-9680 contains 19 contiguous basescomplementary to the bases in the region of NM_(—)174936 from positions3530 through 3548 relative to the start of the mRNA. A set of siRNAmolecules was designed to each unique 19mer of the subset of thetranscript sequence defined by 10 bases upstream from the 5′ end to 10bases downstream from the 3′ end of the target region of AD-9680. Withrespect to the NM_(—)174936.2 transcript, the first base at the 5′position of the sense oligo 19mer extends from positions 3520 topositions 3558 (Tables 7 and 8).

Synthesis of PCSK9 Tiling Sequences:

PCSK9 sequences were synthesized on MerMade 192 synthesizer. Two sets ofsequences were made. The first set contained no chemical modifications(unmodified) and a second set was made with endolight chemicalmodifications. In sequences containing endolight chemical modification,all pyrimidines (cytosine and uridine) in the sense strand were replacedwith corresponding 2′-O-Methyl bases (2′ O-Methyl C and 2′-O-Methyl U).In the antisense strand, pyrimidines adjacent to (towards 5′ position)ribo A nucleoside were replaced with their corresponding 2-O-Methylnucleosides. A two base dTsdT extension at the 3′ end of both sense andanti sense sequences was introduced. The sequence file was converted toa text file to make it compatible for loading in the MerMade 192synthesis software.

The synthesis of PCSK9 sequences used solid supported oligonucleotidesynthesis using phosphoramidite chemistry. The synthesis of the abovesequences was performed at 1 μm scale in 96 well plates. The amiditesolutions were prepared at 0.1 M concentration and ethyl thio tetrazole(0.6M in Acetonitrile) was used as activator. The synthesized sequenceswere cleaved and deprotected in 96 well plates, using methylamine in thefirst step and Fluoride ion in the second step. The crude sequences thusobtained were precipitated using acetone: ethanol mix and the pelletwere re-suspended in 0.2M sodium acetate buffer. Samples from eachsequence were analyzed by LC-MS and the resulting mass data confirmedthe identity of the sequences. A selected set of samples were alsoanalyzed by IEX chromatography. All sequences were purified on AKTAexplorer purification system using Source 15Q column. A single peakcorresponding to the full length sequence was collected in the eluentand was subsequently analyzed for purity by ion exchange chromatography.The purified sequences were desalted on a Sephadex G25 column using AKTApurifier. The desalted PCSK9 sequences were analyzed for concentrationand purity. The single strands were then submitted for annealing.

In Vitro Screening of PCSK9 Tiling siRNAs:

Cell Culture and Transfection:

Hela cells (ATCC, Manassas, Va.) were grown to near confluence at 37° C.in an atmosphere of 5% CO₂ in Eagle's Minimum Essential Medium (EMEM,ATCC) supplemented with 10% FBS, streptomycin, and glutamine (ATCC)before being released from the plate by trypsinization. Reversetransfection was carried out by adding 5 μl of Opti-MEM to 5 μl of siRNAduplexes per well into a 96-well plate along with 10 μl of Opti-MEM plus0.2 μl of Lipofectamine RNAiMax per well (Invitrogen, Carlsbad Calif.cat #13778-150) and incubated at room temperature for 15 minutes. 80 μlof complete growth media without antibiotic containing 2.0×10⁴ Helacells were then added. Cells were incubated for 24 hours prior to RNApurification. Experiments were performed at 0.1 or 10 nM final duplexconcentration. For dose response screens, HeLa cells were transfectedwith siRNAs over seven, ten-fold serial dilutions from 1 nM to 1 fM.

Total RNA was isolated using MagMAX-96 Total RNA Isolation Kit (AppliedBiosystem, Forer City Calif., part #: AM1830). Cells were harvested andlysed in 140 μl of Lysis/Binding Solution then mixed for 1 minute at 850rpm using and Eppendorf Thermomixer (the mixing speed was the samethroughout the process). Twenty micro liters of magnetic beads andLysis/Binding Enhancer mixture were added into cell-lysate and mixed for5 minutes. Magnetic beads were captured using magnetic stand and thesupernatant was removed without disturbing the beads. After removingsupernatant, magnetic beads were washed with Wash Solution 1(isopropanol added) and mixed for 1 minute. Beads were capture again andsupernatant removed. Beads were then washed with 150 μl Wash Solution 2(Ethanol added), captured and supernatant was removed. 50 μl of DNasemixture (MagMax turbo DNase Buffer and Turbo DNase) was then added tothe beads and they were mixed for 10 to 15 minutes. After mixing, 100 μlof RNA Rebinding Solution was added and mixed for 3 minutes. Supernatantwas removed and magnetic beads were washed again with 150 μl WashSolution 2 and mixed for 1 minute and supernatant was removedcompletely. The magnetic beads were mixed for 2 minutes to dry beforeRNA was eluted with 50 μl of water.

cDNA was synthesized using ABI High capacity cDNA reverse transcriptionkit (Applied Biosystems, Foster City, Calif., Cat #4368813). A mastermix of 2 μl 10× Buffer, 0.8 μl 25× dNTPs, 2 μl Random primers, 1 μlReverse Transcriptase, 1 μl RNase inhibitor and 3.2 μl of H2O perreaction were added into 10 μl total RNA. cDNA was generated using aBio-Rad C-1000 or S-1000 thermal cycler (Hercules, Calif.) through thefollowing steps: 25° C. 10 min, 37° C. 120 min, 85° C. 5 sec, 4° C.hold.

Real time PCR was performed as follows. 2 μl of cDNA were added to amaster mix containing 1 μl GAPDH TaqMan Probe (Applied Biosystems Cat#4326317E), 1 μl PCSK9 TaqMan probe (Applied Biosystems cat#HS03037355_M1) and 10 μl Roche Probes Master Mix (Roche Cat#04887301001) per well in a LightCycler 480 384 well plate (Roche cat#0472974001). Real time PCR was done in a LightCycler 480 Real Time PCRmachine (Roche). Each duplex was tested in two independent transfectionsand each transfections was assayed in duplicate.

Real time data were analyzed using the ΔΔ Ct method. Each sample wasnormalized to GAPDH expression and knockdown was assessed relative tocells transfected with the non-targeting duplex AD-1955. IC50s weredefined using a 4 parameter fit model in XLfit.

The data for the single dose experiments are shown in Table 9. Data areexpressed as the percent of message remaining relative to cells targetedwith control AD-1955.

The data for the dose response screen is shown in Table 10. Data areexpressed as dose in pM that results in 50% inhibition relative toAD-1955. Each dose response was repeated twice (Rep1 and Rep2). Averageof the IC50s generated in the two dose response screens is shown.

The average IC50 for siRNA flanking AD-9680 was plotted vs. the startingposition of the target region in the human PCSK9 transcript FIG. 25.

Thus, targeting nucleotide region 3520-3555 of PCSK9 with an RNAi agentis highly effective at inhibiting PCSK9.

Example 19 ApoE3-Based Reconstituted HDL Complexed with dsRNAs TargetingPCSK9

C57BL6 mice were administered 30 mg/kg rEHDL/chol-siPCSK9 by intravenousadministration (tail vein injection) in a single bolus dose.

Chol-siPCSK9 (dsRNA Duplex AD-20583) has the following sequence:

Sense: (SEQ ID NO: 1729) GccuGGAGuuuAuucGGAAdTsdTL10 Antisense:(SEQ ID NO: 1730) PuUfcCfgAfaUfaAfaCfuCfcAfgGfcdTsdT

The structure of L10 is:

After injection, mice were fasted overnight (˜14 hours), and thensacrificed at 48 h post-injection. mRNA levels from liver weredetermined by bDNA assay, and normalized to GAPDH mRNA levels.

Results

The results of the bDNA assays are shown in FIG. 26, which indicate thatthere was a significant reduction in PCSK9 following administration ofrEHDL/chol-siPCSK9, but not following administration of uncomplexedsiRNAs (chol-siPCSK9). rEHDL/chol-siPCSK9 decreased PCSK9 mRNA levels byabout 80%.

Example 20 LNP-11 Formulated siRNA in Non-Human Primates (NHPs)

An siRNA targeting PCSK9 (AD-9680) was formulated in a LNP-11formulation (described herein) and administered to cynomologous monkeys.Control was AD-1955. The lipid formulated siRNAs were administered via a30 minute infusion on day 1 at dosages of 0.03, 0.1, 0.3, and 1.0 mg/kg.Control was administered at 1.0 mg/kg. On day 3, liver biopsies wereperformed for measurement of PCSK9 transcript. Blood samples werecollected on days −3, −1, 3, 4, 5, 7, 9, 11, 12, 15, 22, 30, and 37 andPCSK9 protein levels and LDLc numbers and HDLc numbers were determined.

The results are shown in FIG. 27A, FIG. 27B, and FIG. 27C.

As shown in FIG. 27A and FIG. 27B, administration resulted in a rapidand durable dose dependent reduction in PCSK9 protein levels andresulted in >50% reduction in LDLc (LDL cholesterol) levels. Theseeffects were very potent with ED50 dose levels between 30 and 100micrograms per kilogram. As shown in FIG. 27C, administration resultedin no change in HDLc levels.

Example 21 Dose Response in Rats with LNP-09 Formulated PCSK9 dsRNA

The dsRNA AD-10792 (targeting rate PCSK9) was encapsulated in a XTCcontaining formulation, e.g., a LNP09 formulation. LNP09 formulation wasXTC/DSPC/Cholesterol/PEG-DMG at a % mol ratio of 50/10/38.5/1.5 and alipid:siRNA ratio of 10:1.

Formulations were injected via tail vein, single dose (DRC) into rats.Livers and plasma were harvested 72 hours post-injection (5 animals pergroup). PCSK9 transcript levels were measured via bDNA in liversprepared as manufacturer's protocol. GAPDH transcript levels were alsomeasured and the PCSK9 to GAPDH ratios were normalized to those of PBScontrol and graphed. Total cholesterol was measured in serum usingcholesterol kit from WAKO TX.

The results are shown in FIG. 29. With this formulation PCSK9 silencingand total cholesterol lowering in rats was achieved at doses <0.1 mg/kg.The ED₅₀ for was 0.2 mg/kg for lowering PCSK9 mRNA and 0.2 mg/kg and0.08 for lowering serum cholesterol.

Example 22 Treatment of Transgenic Mice with LNP-09 Formulated PCSK9dsRNA

Transgenic mice that overexpress human CETP and ApoB 100 (CETP/ApoBdouble humanized transgenic mice, Taconic Labs) more closely mimic theLDL/HDL ratios found in man.

CETP/ApoB double humanized transgenic mice were purchased from Taconiclabs. Animals were injected through tail vein (single injection) of 5mg/kg of LNP09 formulated AD-10792 (standard formulation procedure), orAD-1955 Luciferase control (4 animals per group). Livers and plasma wereharvested 72 hours post-injection (5 animals per group) and liver PCSK9mRNA, LDL particle, and HDL particle number were determined.

PCSK9 transcript levels were measured via bDNA in livers preparedaccording to manufacturer's protocol. GAPDH transcript levels were alsomeasured and the PCSK9 to GAPDH ratios were graphed, normalized to thoseof PBS control. LDL and HDL particle numbers/concentration were measuredby NMR (Liposciences Inc.) based on their SOP.

The results are shown in FIG. 30. Silencing of PCSK9 lowered LDLparticle concentrations ˜90%, while HDL levels were modestly lower (ascompared to those treated animals treated with PBS controls). Thisdemonstrates significant lowering of PCSK9 levels with subsequent LDLclowering in these animals.

Example 23 Inhibition of PCSK9 Expression in Humans

A human subject is treated with a lipid formulated dsRNA targeted to aPCSK9 gene, described herein, to inhibit expression of the PCSK9 geneand lower cholesterol levels for an extended period of time following asingle dose. In one embodiment, the lipid formulated dsRNA includes thelipid MC3.

A subject in need of treatment is selected or identified. The subjectcan be in need of LDL lowering, LDL lowering without lowering of HDL,ApoB lowering, or total cholesterol lowering. The identification of thesubject can occur in a clinical setting, or elsewhere, e.g., in thesubject's home through the subject's own use of a self-testing kit.

At time zero, a suitable first dose of an anti-PCSK9 siRNA issubcutaneously administered to the subject. The dsRNA is formulated asdescribed herein. After a period of time following the first dose, e.g.,7 days, 14 days, and 21 days, the subject's condition is evaluated,e.g., by measuring LDL, ApoB, and/or total cholesterol levels. Thismeasurement can be accompanied by a measurement of PCSK9 expression insaid subject, and/or the products of the successful siRNA-targeting ofPCSK9 mRNA. Other relevant criteria can also be measured. The number andstrength of doses are adjusted according to the subject's needs.

After treatment, the subject's LDL, ApoB, or total cholesterol levelsare lowered relative to the levels existing prior to the treatment, orrelative to the levels measured in a similarly afflicted but untreatedsubject.

Those skilled in the art are familiar with methods and compositions inaddition to those specifically set out in the present disclosure whichwill allow them to practice this invention to the full scope of theclaims hereinafter appended.

TABLE 1a dsRNA sequences targeted to PCSK9 position in human access. SEQSEQ # NM_ ID ID Duplex 174936 Sense strand sequence (5'-3')¹ NO:Antisense-strand sequence (5'-3')¹ NO: name    2-20AGCGACGUCGAGGCGCUCATT 1 UGAGCGCCUCGACGUCGCUTT 2 AD- 15220   15-33CGCUCAUGGUUGCAGGCGGTT 3 CCGCCUGCAACCAUGAGCGTT 4 AD- 15275   16-34GCUCAUGGUUGCAGGCGGGTT 5 CCCGCCUGCAACCAUGAGCTT 6 AD- 15301   30-48GCGGGCGCCGCCGUUCAGUTT 7 ACUGAACGGCGGCGCCCGCTT 8 AD- 15276   31-49CGGGCGCCGCCGUUCAGUUTT 9 AACUGAACGGCGGCGCCCGTT 10 AD- 15302   32-50GGGCGCCGCCGUUCAGUUCTT 11 GAACUGAACGGCGGCGCCCTT 12 AD- 15303   40-58CCGUUCAGUUCAGGGUCUGTT 13 CAGACCCUGAACUGAACGGTT 14 AD- 15221   43-61UUCAGUUCAGGGUCUGAGCTT 15 GCUCAGACCCUGAACUGAATT 16 AD- 15413   82-100GUGAGACUGGCUCGGGCGGTT 17 CCGCCCGAGCCAGUCUCACTT 18 AD- 15304  100-118GGCCGGGACGCGUCGUUGCTT 19 GCAACGACGCGUCCCGGCCTT 20 AD- 15305  101-119GCCGGGACGCGUCGUUGCATT 21 UGCAACGACGCGUCCCGGCTT 22 AD- 15306  102-120CCGGGACGCGUCGUUGCAGTT 23 CUGCAACGACGCGUCCCGGTT 24 AD- 15307  105-123GGACGCGUCGUUGCAGCAGTT 25 CUGCUGCAACGACGCGUCCTT 26 AD- 15277  135-153UCCCAGCCAGGAUUCCGCGTsT 27 CGCGGAAUCCUGGCUGGGATsT 28 AD- 9526  135-153ucccAGccAGGAuuccGcGTsT 29 CGCGGAAUCCUGGCUGGGATsT 30 AD- 9652  136-154CCCAGCCAGGAUUCCGCGCTsT 31 GCGCGGAAUCCUGGCUGGGTsT 32 AD- 9519  136-154cccAGccAGGAuuccGcGcTsT 33 GCGCGGAAUCCUGGCUGGGTsT 34 AD- 9645  138-156CAGCCAGGAUUCCGCGCGCTsT 35 GCGCGCGGAAUCCUGGCUGTsT 36 AD- 9523  138-156cAGccAGGAuuccGcGcGcTsT 37 GCGCGCGGAAUCCUGGCUGTsT 38 AD- 9649  185-203AGCUCCUGCACAGUCCUCCTsT 39 GGAGGACUGUGCAGGAGCUTsT 40 AD- 9569  185-203AGcuccuGcAcAGuccuccTsT 41 GGAGGACUGUGcAGGAGCUTsT 42 AD- 9695  205-223CACCGCAAGGCUCAAGGCGTT 43 CGCCUUGAGCCUUGCGGUGTT 44 AD- 15222  208-226CGCAAGGCUCAAGGCGCCGTT 45 CGGCGCCUUGAGCCUUGCGTT 46 AD- 15278  210-228CAAGGCUCAAGGCGCCGCCTT 47 GGCGGCGCCUUGAGCCUUGTT 48 AD- 15178  232-250GUGGACCGCGCACGGCCUCTT 49 GAGGCCGUGCGCGGUCCACTT 50 AD- 15308  233-251UGGACCGCGCACGGCCUCUTT 51 AGAGGCCGUGCGCGGUCCATT 52 AD- 15223  234-252GGACCGCGCACGGCCUCUATT 53 UAGAGGCCGUGCGCGGUCCTT 54 AD- 15309  235-253GACCGCGCACGGCCUCUAGTT 55 CUAGAGGCCGUGCGCGGUCTT 56 AD- 15279  236-254ACCGCGCACGGCCUCUAGGTT 57 CCUAGAGGCCGUGCGCGGUTT 58 AD- 15194  237-255CCGCGCACGGCCUCUAGGUTT 59 ACCUAGAGGCCGUGCGCGGTT 60 AD- 15310  238-256CGCGCACGGCCUCUAGGUCTT 61 GACCUAGAGGCCGUGCGCGTT 62 AD- 15311  239-257GCGCACGGCCUCUAGGUCUTT 63 AGACCUAGAGGCCGUGCGCTT 64 AD- 15392  240-258CGCACGGCCUCUAGGUCUCTT 65 GAGACCUAGAGGCCGUGCGTT 66 AD- 15312  248-266CUCUAGGUCUCCUCGCCAGTT 67 CUGGCGAGGAGACCUAGAGTT 68 AD- 15313  249-267UCUAGGUCUCCUCGCCAGGTT 69 CCUGGCGAGGAGACCUAGATT 70 AD- 15280  250-268CUAGGUCUCCUCGCCAGGATT 71 UCCUGGCGAGGAGACCUAGTT 72 AD- 15267  252-270AGGUCUCCUCGCCAGGACATT 73 UGUCCUGGCGAGGAGACCUTT 74 AD- 15314  258-276CCUCGCCAGGACAGCAACCTT 75 GGUUGCUGUCCUGGCGAGGTT 76 AD- 15315  300-318CGUCAGCUCCAGGCGGUCCTsT 77 GGACCGCCUGGAGCUGACGTsT 78 AD- 9624  300-318cGucAGcuccAGGcGGuccTsT 79 GGACCGCCUGGAGCUGACGTsT 80 AD- 9750  301-319GUCAGCUCCAGGCGGUCCUTsT 81 AGGACCGCCUGGAGCUGACTsT 82 AD- 9623  301-319GucAGcuccAGGcGGuccuTsT 83 AGGACCGCCUGGAGCUGACTsT 84 AD- 9749  370-388GGCGCCCGUGCGCAGGAGGTT 85 CCUCCUGCGCACGGGCGCCTT 86 AD- 15384  408-426GGAGCUGGUGCUAGCCUUGTsT 87 CAAGGCUAGCACCAGCUCCTsT 88 AD- 9607  408-426GGAGcuGGuGcuAGccuuGTsT 89 cAAGGCuAGcACcAGCUCCTsT 90 AD- 9733  411-429GCUGGUGCUAGCCUUGCGUTsT 91 ACGCAAGGCUAGCACCAGCTsT 92 AD- 9524  411-429GcuGGuGcuAGccuuGcGuTsT 93 ACGcAAGGCuAGcACcAGCTsT 94 AD- 9650  412-430CUGGUGCUAGCCUUGCGUUTsT 95 AACGCAAGGCUAGCACCAGTsT 96 AD- 9520  412-430CUGGUGCUAGCCUUGCGUUTsT 97 AACGCAAGGCUAGCACCAGTsT 98 AD- 9520  412-430cuGGuGcuAGccuuGcGuuTsT 99 AACGcAAGGCuAGcACcAGTsT 100 AD- 9646  416-434UGCUAGCCUUGCGUUCCGATsT 101 UCGGAACGCAAGGCUAGCATsT 102 AD- 9608  416-434uGcuAGccuuGcGuuccGATsT 103 UCGGAACGcAAGGCuAGcATsT 104 AD- 9734  419-437UAGCCUUGCGUUCCGAGGATsT 105 UCCUCGGAACGCAAGGCUATsT 106 AD- 9546  419-437uAGccuuGcGuuccGAGGATsT 107 UCCUCGGAACGcAAGGCuATsT 108 AD- 9672  439-457GACGGCCUGGCCGAAGCACTT 109 GUGCUUCGGCCAGGCCGUCTT 110 AD- 15385  447-465GGCCGAAGCACCCGAGCACTT 111 GUGCUCGGGUGCUUCGGCCTT 112 AD- 15393  448-466GCCGAAGCACCCGAGCACGTT 113 CGUGCUCGGGUGCUUCGGCTT 114 AD- 15316  449-467CCGAAGCACCCGAGCACGGTT 115 CCGUGCUCGGGUGCUUCGGTT 116 AD- 15317  458-476CCGAGCACGGAACCACAGCTT 117 GCUGUGGUUCCGUGCUCGGTT 118 AD- 15318  484-502CACCGCUGCGCCAAGGAUCTT 119 GAUCCUUGGCGCAGCGGUGTT 120 AD- 15195  486-504CCGCUGCGCCAAGGAUCCGTT 121 CGGAUCCUUGGCGCAGCGGTT 122 AD- 15224  487-505CGCUGCGCCAAGGAUCCGUTT 123 ACGGAUCCUUGGCGCAGCGTT 124 AD- 15188  489-507CUGCGCCAAGGAUCCGUGGTT 125 CCACGGAUCCUUGGCGCAGTT 126 AD- 15225  500-518AUCCGUGGAGGUUGCCUGGTT 127 CCAGGCAACCUCCACGGAUTT 128 AD- 15281  509-527GGUUGCCUGGCACCUACGUTT 129 ACGUAGGUGCCAGGCAACCTT 130 AD- 15282  542-560AGGAGACCCACCUCUCGCATT 131 UGCGAGAGGUGGGUCUCCUTT 132 AD- 15319  543-561GGAGACCCACCUCUCGCAGTT 133 CUGCGAGAGGUGGGUCUCCTT 134 AD- 15226  544-562GAGACCCACCUCUCGCAGUTT 135 ACUGCGAGAGGUGGGUCUCTT 136 AD- 15271  549-567CCACCUCUCGCAGUCAGAGTT 137 CUCUGACUGCGAGAGGUGGTT 138 AD- 15283  552-570CCUCUCGCAGUCAGAGCGCTT 139 GCGCUCUGACUGCGAGAGGTT 140 AD- 15284  553-571CUCUCGCAGUCAGAGCGCATT 141 UGCGCUCUGACUGCGAGAGTT 142 AD- 15189  554-572UCUCGCAGUCAGAGCGCACTT 143 GUGCGCUCUGACUGCGAGATT 144 AD- 15227  555-573CUCGCAGUCAGAGCGCACUTsT 145 AGUGCGCUCUGACUGCGAGTsT 146 AD- 9547  555-573cucGcAGucAGAGcGcAcuTsT 147 AGUGCGCUCUGACUGCGAGTsT 148 AD- 9673  558-576GCAGUCAGAGCGCACUGCCTsT 149 GGCAGUGCGCUCUGACUGCTsT 150 AD- 9548  558-576GcAGucAGAGcGcAcuGccTsT 151 GGcAGUGCGCUCUGACUGCTsT 152 AD- 9674  606-624GGGAUACCUCACCAAGAUCTsT 153 GAUCUUGGUGAGGUAUCCCTsT 154 AD- 9529  606-624GGGAuAccucAccAAGAucTsT 155 GAUCUUGGUGAGGuAUCCCTsT 156 AD- 9655  659-677UGGUGAAGAUGAGUGGCGATsT 157 UCGCCACUCAUCUUCACCATsT 158 AD- 9605  659-677uGGuGAAGAuGAGuGGcGATsT 159 UCGCcACUcAUCUUcACcATsT 160 AD- 9731  663-681GAAGAUGAGUGGCGACCUGTsT 161 CAGGUCGCCACUCAUCUUCTsT 162 AD- 9596  663-681GAAGAuGAGuGGcGAccuGTsT 163 cAGGUCGCcACUcAUCUUCTsT 164 AD- 9722  704-722CCCAUGUCGACUACAUCGATsT 165 UCGAUGUAGUCGACAUGGGTsT 166 AD- 9583  704-722cccAuGucGAcuAcAucGATsT 167 UCGAUGuAGUCGAcAUGGGTsT 168 AD- 9709  718-736AUCGAGGAGGACUCCUCUGTsT 169 CAGAGGAGUCCUCCUCGAUTsT 170 AD- 9579  718-736AucGAGGAGGAcuccucuGTsT 171 cAGAGGAGUCCUCCUCGAUTsT 172 AD- 9705  758-776GGAACCUGGAGCGGAUUACTT 173 GUAAUCCGCUCCAGGUUCCTT 174 AD- 15394  759-777GAACCUGGAGCGGAUUACCTT 175 GGUAAUCCGCUCCAGGUUCTT 176 AD- 15196  760-778AACCUGGAGCGGAUUACCCTT 177 GGGUAAUCCGCUCCAGGUUTT 178 AD- 15197  777-795CCCUCCACGGUACCGGGCGTT 179 CGCCCGGUACCGUGGAGGGTT 180 AD- 15198  782-800CACGGUACCGGGCGGAUGATsT 181 UCAUCCGCCCGGUACCGUGTsT 182 AD- 9609  782-800cAcGGuAccGGGcGGAuGATsT 183 UcAUCCGCCCGGuACCGUGTsT 184 AD- 9735  783-801ACGGUACCGGGCGGAUGAATsT 185 UUCAUCCGCCCGGUACCGUTsT 186 AD- 9537  783-801AcGGuAccGGGcGGAuGAATsT 187 UUcAUCCGCCCGGuACCGUTsT 188 AD- 9663  784-802CGGUACCGGGCGGAUGAAUTsT 189 AUUCAUCCGCCCGGUACCGTsT 190 AD- 9528  784-802cGGuAccGGGcGGAuGAAuTsT 191 AUUcAUCCGCCCGGuACCGTsT 192 AD- 9654  785-803GGUACCGGGCGGAUGAAUATsT 193 UAUUCAUCCGCCCGGUACCTsT 194 AD- 9515  785-803GGuAccGGGcGGAuGAAuATsT 195 uAUUcAUCCGCCCGGuACCTsT 196 AD- 9641  786-804GUACCGGGCGGAUGAAUACTsT 197 GUAUUCAUCCGCCCGGUACTsT 198 AD- 9514  786-804GuAccGGGcGGAuGAAuAcTsT 199 GuAUUcAUCCGCCCGGuACTsT 200 AD- 9640  788-806ACCGGGCGGAUGAAUACCATsT 201 UGGUAUUCAUCCGCCCGGUTsT 202 AD- 9530  788-806AccGGGcGGAuGAAuAccATsT 203 UGGuAUUcAUCCGCCCGGUTsT 204 AD- 9656  789-807CCGGGCGGAUGAAUACCAGTsT 205 CUGGUAUUCAUCCGCCCGGTsT 206 AD- 9538  789-807ccGGGcGGAuGAAuAccAGTsT 207 CUGGuAUUcAUCCGCCCGGTsT 208 AD- 9664  825-843CCUGGUGGAGGUGUAUCUCTsT 209 GAGAUACACCUCCACCAGGTsT 210 AD- 9598  825-843ccuGGuGGAGGuGuAucucTsT 211 GAGAuAcACCUCcACcAGGTsT 212 AD- 9724  826-844CUGGUGGAGGUGUAUCUCCTsT 213 GGAGAUACACCUCCACCAGTsT 214 AD- 9625  826-844cuGGuGGAGGuGuAucuccTsT 215 GGAGAuAcACCUCcACcAGTsT 216 AD- 9751  827-845UGGUGGAGGUGUAUCUCCUTsT 217 AGGAGAUACACCUCCACCATsT 218 AD- 9556  827-845uGGuGGAGGuGuAucuccuTsT 219 AGGAGAuAcACCUCcACcATsT 220 AD- 9682  828-846GGUGGAGGUGUAUCUCCUATsT 221 UAGGAGAUACACCUCCACCTsT 222 AD- 9539  828-846GGuGGAGGuGuAucuccuATsT 223 uAGGAGAuAcACCUCcACCTsT 224 AD- 9665  831-849GGAGGUGUAUCUCCUAGACTsT 225 GUCUAGGAGAUACACCUCCTsT 226 AD- 9517  831-849GGAGGuGuAucuccuAGAcTsT 227 GUCuAGGAGAuAcACCUCCTsT 228 AD- 9643  833-851AGGUGUAUCUCCUAGACACTsT 229 GUGUCUAGGAGAUACACCUTsT 230 AD- 9610  833-851AGGuGuAucuccuAGAcAcTsT 231 GUGUCuAGGAGAuAcACCUTsT 232 AD- 9736  833-851AfgGfuGfuAfuCfuCfcUfaGfaCfaC 233 p- 234 AD- fTsTgUfgUfcUfaGfgAfgAfuAfcAfcCfuTsT 14681  833-851AGGUfGUfAUfCfUfCfCfUfAGACfAC 235 GUfGUfCfUfAGGAGAUfACfACfCfUfTsT 236 AD-fTsT 14691  833-851 AgGuGuAuCuCcUaGaCaCTsT 237 p- 238 AD-gUfgUfcUfaGfgAfgAfuAfcAfcCfuTsT 14701  833-851 AgGuGuAuCuCcUaGaCaCTsT239 GUfGUfCfUfAGGAGAUfACfACfCfUfTsT 240 AD- 14711  833-851AfgGfuGfuAfuCfuCfcUfaGfaCfaC 241 GUGUCuaGGagAUACAccuTsT 242 AD- fTsT14721  833-851 AGGUfGUfAUfCfUfCfCfUfAGACfAC 243 GUGUCuaGGagAUACAccuTsT244 AD- fTsT 14731  833-851 AgGuGuAuCuCcUaGaCaCTsT 245GUGUCuaGGagAUACAccuTsT 246 AD- 14741  833-851GfcAfcCfcUfcAfuAfgGfcCfuGfgA 247 p- 248 AD- fTsTuCfcAfgGfcCfuAfuGfaGfgGfuGfcTsT 15087  833-851GCfACfCfCfUfCfAUfAGGCfCfUfGG 249 UfCfCfAGGCfCfUfAUfGAGGGUfGCfTsT 250 AD-ATsT 15097  833-851 GcAcCcUcAuAgGcCuGgATsT 251 p- 252 AD-uCfcAfgGfcCfuAfuGfaGfgGfuGfcTsT 15107  833-851 GcAcCcUcAuAgGcCuGgATsT253 UfCfCfAGGCfCfUfAUfGAGGGUfGCfTsT 254 AD- 15117  833-851GfcAfcCfcUfcAfuAfgGfcCfuGfgA 255 UCCAGgcCUauGAGGGugcTsT 256 AD- fTsT15127  833-851 GCfACfCfCfUfCfAUfAGGCfCfUfGG 257 UCCAGgcCUauGAGGGugcTsT258 AD- ATsT 15137  833-851 GcAcCcUcAuAgGcCuGgATsT 259UCCAGgcCUauGAGGGugcTsT 260 AD- 15147  836-854 UGUAUCUCCUAGACACCAGTsT 261CUGGUGUCUAGGAGAUACATsT 262 AD- 9516  836-854 uGuAucuccuAGAcAccAGTsT 263CUGGUGUCuAGGAGAuAcATsT 264 AD- 9642  840-858 UCUCCUAGACACCAGCAUATsT 265UAUGCUGGUGUCUAGGAGATsT 266 AD- 9562  840-858 ucuccuAGAcAccAGcAuATsT 267uAUGCUGGUGUCuAGGAGATsT 268 AD- 9688  840-858UfcUfcCfuAfgAfcAfcCfaGfcAfuA 269 p- 270 AD- fTsTuAfuGfcUfgGfuGfuCfuAfgGfaGfaTsT 14677  840-858UfCfUfCfCfUfAGACfACfCfAGCfAU 271 UfAUfGCfUfGGUfGUfCfUfAGGAGATsT 272 AD-fATsT 14687  840-858 UcUcCuAgAcAcCaGcAuATsT 273 p- 274 AD-uAfuGfcUfgGfuGfuCfuAfgGfaGfaTsT 14697  840-858 UcUcCuAgAcAcCaGcAuATsT275 UfAUfGCfUfGGUfGUfCfUfAGGAGATsT 276 AD- 14707  840-858UfcUfcCfuAafAfcAfcCfaGfcAfuA 277 UAUGCugGUguCUAGGagaTsT 278 AD- fTsT14717  840-858 UfCfUfCfCfUfAGACfACfCfAGCfAU 279 UAUGCugGUguCUAGGagaTsT280 AD- fATsT 14727  840-858 UcUcCuAgAcAcCaGcAuATsT 281UAUGCugGUguCUAGGagaTsT 282 AD- 14737  840-858AfgGfcCfuGfgAfgUfuUfaUfuCfgG 283 p- 284 AD- fTsTcCfgAfaUfaAfaCfuCfcAfgGfcCfuTsT 15083  840-858AGGCfCfUfGGAGUfUfUfAUfUfCfGG 285 CfCfGAAUfAAACfUfCfCfAGGCfCfUfTs 286 AD-TsT T 15093  840-858 AgGcCuGgAgUuUaUuCgGTsT 287 p- 288 AD-cCfgAfaUfaAfaCfuCfcAfgGfcCfuTsT 15103  840-858 AgGcCuGgAgUuUaUuCgGTsT289 CfCfGAAUfAAACfUfCfCfAGGCfCfUfTs 290 AD- T 15113  840-858AfgGfcCfuGfgAfgUfuUfaUfuCfgG 291 CCGAAuaAAcuCCAGGccuTsT 292 AD- fTsT15123  840-858 AGGCfCfUfGGAGUfUfUfAUfUfCfGG 293 CCGAAuaAAcuCCAGGccuTsT294 AD- TsT 15133  840-858 AgGcCuGgAgUuUaUuCgGTsT 295CCGAAuaAAcuCCAGGccuTsT 296 AD- 15143  841-859 CUCCUAGACACCAGCAUACTsT 297GUAUGCUGGUGUCUAGGAGTsT 298 AD- 9521  841-859 cuccuAGAcAccAGcAuAcTsT 299GuAUGCUGGUGUCuAGGAGTsT 300 AD- 9647  842-860 UCCUAGACACCAGCAUACATsT 301UGUAUGCUGGUGUCUAGGATsT 302 AD- 9611  842-860 uccuAGAcAccAGcAuAcATsT 303UGuAUGCUGGUGUCuAGGATsT 304 AD- 9737  843-861 CCUAGACACCAGCAUACAGTsT 305CUGUAUGCUGGUGUCUAGGTsT 306 AD- 9592  843-861 ccuAGAcAccAGcAuAcAGTsT 307CUGuAUGCUGGUGUCuAGGTsT 308 AD- 9718  847-865 GACACCAGCAUACAGAGUGTsT 309CACUCUGUAUGCUGGUGUCTsT 310 AD- 9561  847-865 GAcAccAGcAuAcAGAGuGTsT 311cACUCUGuAUGCUGGUGUCTsT 312 AD- 9687  855-873 CAUACAGAGUGACCACCGGTsT 313CCGGUGGUCACUCUGUAUGTsT 314 AD- 9636  855-873 cAuAcAGAGuGAccAccGGTsT 315CCGGUGGUcACUCUGuAUGTsT 316 AD- 9762  860-878 AGAGUGACCACCGGGAAAUTsT 317AUUUCCCGGUGGUCACUCUTsT 318 AD- 9540  860-878 AGAGuGAccAccGGGAAAuTsT 319AUUUCCCGGUGGUcACUCUTsT 320 AD- 9666  861-879 GAGUGACCACCGGGAAAUCTsT 321GAUUUCCCGGUGGUCACUCTsT 322 AD- 9535  861-879 GAGuGAccAccGGGAAAucTsT 323GAUUUCCCGGUGGUcACUCTsT 324 AD- 9661  863-881 GUGACCACCGGGAAAUCGATsT 325UCGAUUUCCCGGUGGUCACTsT 326 AD- 9559  863-881 GuGAccAccGGGAAAucGATsT 327UCGAUUUCCCGGUGGUcACTsT 328 AD- 9685  865-883 GACCACCGGGAAAUCGAGGTsT 329CCUCGAUUUCCCGGUGGUCTsT 330 AD- 9533  865-883 GAccAccGGGAAAucGAGGTsT 331CCUCGAUUUCCCGGUGGUCTsT 332 AD- 9659  866-884 ACCACCGGGAAAUCGAGGGTsT 333CCCUCGAUUUCCCGGUGGUTsT 334 AD- 9612  866-884 AccAccGGGAAAucGAGGGTsT 335CCCUCGAUUUCCCGGUGGUTsT 336 AD- 9738  867-885 CCACCGGGAAAUCGAGGGCTsT 337GCCCUCGAUUUCCCGGUGGTsT 338 AD- 9557  867-885 ccAccGGGAAAucGAGGGcTsT 339GCCCUCGAUUUCCCGGUGGTsT 340 AD- 9683  875-893 AAAUCGAGGGCAGGGUCAUTsT 341AUGACCCUGCCCUCGAUUUTsT 342 AD- 9531  875-893 AAAucGAGGGcAGGGucAuTsT 343AUGACCCUGCCCUCGAUUUTsT 344 AD- 9657  875-893AfaAfuCfgAfgGfgCfaGfgGfuCfaU 345 p- 346 AD- fTsTaUfgAfcCfcUfgCfcCfuCfgAfuUfuTsT 14673  875-893AAAUfCfGAGGGCfAGGGUfCfAUfTsT 347 AUfGACfCfCfUfGCfCfCfUfCfGAUfUfU 348 AD-fTsT 14683  875-893 AaAuCgAgGgCaGgGuCaUTsT 349 p- 350 AD-aUfgAfcCfcUfgCfcCfuCfgAfuUfuTsT 14693  875-893 AaAuCgAgGgCaGgGuCaUTsT351 AUfGACfCfCfUfGCfCfCfUfCfGAUfUfU 352 AD- fTsT 14703  875-893AfaAfuCfgAfgGfgCfaGfgGfuCfaU 353 AUGACccUGccCUCGAuuuTsT 354 AD- fTsT14713  875-893 AAAUfCfGAGGGCfAGGGUfCfAUfTsT 355 AUGACccUGccCUCGAuuuTsT356 AD- 14723  875-893 AaAuCgAgGgCaGgGuCaUTsT 357 AUGACccUGccCUCGAuuuTsT358 AD- 14733  875-893 CfgGfcAfcCfcUfcAfuAfgGfcCfuG 359 p- 360 AD- fTsTcAfgGfcCfuAfuGfaGfgGfuGfcCfgTsT 15079  875-893CfGGCfACfCfCfUfCfAUfAGGCfCfU 361 CfAGGCfCfUfAUfGAGGGUfGCfCfGTsT 362 AD-fGTsT 15089  875-893 CgGcAcCcUcAuAgGcCuGTsT 363 p- 364 AD-cAfgGfcCfuAfuGfaGfgGfuGfcCfgTsT 15099  875-893 CgGcAcCcUcAuAgGcCuGTsT365 CfAGGCfCfUfAUfGAGGGUfGCfCfGTsT 366 AD- 15109  875-893CfgGfcAfcCfcUfcAfuAfgGfcCfuG 367 CAGGCcuAUgaGGGUGccgTsT 368 AD- fTsT15119  875-893 CfGGCfACfCfCfUfCfAUfAGGCfCfU 369 CAGGCcuAUgaGGGUGccgTsT370 AD- fGTsT 15129  875-893 CgGcAcCcUcAuAgGcCuGTsT 371CAGGCcuAUgaGGGUGccgTsT 372 AD- 15139  877-895 AUCGAGGGCAGGGUCAUGGTsT 373CCAUGACCCUGCCCUCGAUTsT 374 AD- 9542  877-895 AucGAGGGcAGGGucAuGGTsT 375CcAUGACCCUGCCCUCGAUTsT 376 AD- 9668  878-896 cGAGGGcAGGGucAuGGucTsT 377GACcAUGACCCUGCCCUCGTsT 378 AD- 9739  880-898 GAGGGCAGGGUCAUGGUCATsT 379UGACCAUGACCCUGCCCUCTsT 380 AD- 9637  880-898 GAGGGcAGGGucAuGGucATsT 381UGACcAUGACCCUGCCCUCTsT 382 AD- 9763  882-900 GGGCAGGGUCAUGGUCACCTsT 383GGUGACCAUGACCCUGCCCTsT 384 AD- 9630  882-900 GGGcAGGGucAuGGucAccTsT 385GGUGACcAUGACCCUGCCCTsT 386 AD- 9756  885-903 CAGGGUCAUGGUCACCGACTsT 387GUCGGUGACCAUGACCCUGTsT 388 AD- 9593  885-903 cAGGGucAuGGucAccGAcTsT 389GUCGGUGACcAUGACCCUGTsT 390 AD- 9719  886-904 AGGGUCAUGGUCACCGACUTsT 391AGUCGGUGACCAUGACCCUTsT 392 AD- 9601  886-904 AGGGucAuGGucAccGAcuTsT 393AGUCGGUGACcAUGACCCUTsT 394 AD- 9727  892-910 AUGGUCACCGACUUCGAGATsT 395UCUCGAAGUCGGUGACCAUTsT 396 AD- 9573  892-910 AuGGucAccGAcuucGAGATsT 397UCUCGAAGUCGGUGACcAUTsT 398 AD- 9699  899-917 CCGACUUCGAGAAUGUGCCTT 399GGCACAUUCUCGAAGUCGGTT 400 AD- 15228  921-939 GGAGGACGGGACCCGCUUCTT 401GAAGCGGGUCCCGUCCUCCTT 402 AD- 15395  993- CAGCGGCCGGGAUGCCGGCTsT 403GCCGGCAUCCCGGCCGCUGTsT 404 AD- 1011 9602  993- cAGcGGccGGGAuGccGGcTsT405 GCCGGcAUCCCGGCCGCUGTsT 406 AD- 1011 9728 1020- GGGUGCCAGCAUGCGCAGCTT407 GCUGCGCAUGCUGGCACCCTT 408 AD- 1038 15386 1038-CCUGCGCGUGCUCAACUGCTsT 409 GCAGUUGAGCACGCGCAGGTsT 410 AD- 1056 95801038- ccuGcGcGuGcucAAcuGcTsT 411 GcAGUUGAGcACGCGcAGGTsT 412 AD- 10569706 1040- UGCGCGUGCUCAACUGCCATsT 413 UGGCAGUUGAGCACGCGCATsT 414 AD-1058 9581 1040- uGcGcGuGcucAAcuGccATsT 415 UGGcAGUUGAGcACGCGcATsT 416AD- 1058 9707 1042- CGCGUGCUCAACUGCCAAGTsT 417 CUUGGCAGUUGAGCACGCGTsT418 AD- 1060 9543 1042- cGcGuGcucAAcuGccAAGTsT 419CUUGGcAGUUGAGcACGCGTsT 420 AD- 1060 9669 1053- CUGCCAAGGGAAGGGCACGTsT421 CGUGCCCUUCCCUUGGCAGTsT 422 AD- 1071 9574 1053-cuGccAAGGGAAGGGcAcGTsT 423 CGUGCCCUUCCCUUGGcAGTsT 424 AD- 1071 97001057- CAAGGGAAGGGCACGGUUATT 425 UAACCGUGCCCUUCCCUUGTT 426 AD- 1075 153201058- AAGGGAAGGGCACGGUUAGTT 427 CUAACCGUGCCCUUCCCUUTT 428 AD- 1076 153211059- AGGGAAGGGCACGGUUAGCTT 429 GCUAACCGUGCCCUUCCCUTT 430 AD- 1077 151991060- GGGAAGGGCACGGUUAGCGTT 431 CGCUAACCGUGCCCUUCCCTT 432 AD- 1078 151671061- GGAAGGGCACGGUUAGCGGTT 433 CCGCUAACCGUGCCCUUCCTT 434 AD- 1079 151641062- GAAGGGCACGGUUAGCGGCTT 435 GCCGCUAACCGUGCCCUUCTT 436 AD- 1080 151661063- AAGGGCACGGUUAGCGGCATT 437 UGCCGCUAACCGUGCCCUUTT 438 AD- 1081 153221064- AGGGCACGGUUAGCGGCACTT 439 GUGCCGCUAACCGUGCCCUTT 440 AD- 1082 152001068- CACGGUUAGCGGCACCCUCTT 441 GAGGGUGCCGCUAACCGUGTT 442 AD- 1086 152131069- ACGGUUAGCGGCACCCUCATT 443 UGAGGGUGCCGCUAACCGUTT 444 AD- 1087151229 1072- GUUAGCGGCACCCUCAUAGTT 445 CUAUGAGGGUGCCGCUAACTT 446 AD-1090 152215 1073- UUAGCGGCACCCUCAUAGGTT 447 CCUAUGAGGGUGCCGCUAATT 448AD- 1091 15214 1076- GCGGCACCCUCAUAGGCCUTsT 449 AGGCCUAUGAGGGUGCCGCTsT450 AD- 1094 9315 1079- GCACCCUCAUAGGCCUGGATsT 451UCCAGGCCUAUGAGGGUGCTsT 452 AD- 1097 9326 1085- UCAUAGGCCUGGAGUUUAUTsT453 AUAAACUCCAGGCCUAUGATsT 454 AD- 1103 9318 1090-GGCCUGGAGUUUAUUCGGATsT 455 UCCGAAUAAACUCCAGGCCTsT 456 AD- 1108 93231091- GCCUGGAGUUUAUUCGGAATsT 457 UUCCGAAUAAACUCCAGGCTsT 458 AD- 11099314 1091- GccuGGAGuuuAuucGGAATsT 459 UUCCGAAuAAACUCcAGGCTsT 460 AD-1109 10792 1091- GccuGGAGuuuAuucGGAATsT 461 UUCCGAAUAACUCCAGGCTsT 462AD- 1109 10796 1093- CUGGAGUUUAUUCGGAAAATsT 463 UUUUCCGAAUAAACUCCAGTsT464 AD- 1111 9638 1093- cuGGAGuuuAuucGGAAAATsT 465UUUUCCGAAuAAACUCcAGTsT 466 AD- 1111 9764 1095- GGAGUUUAUUCGGAAAAGCTsT467 GCUUUUCCGAAUAAACUCCTsT 468 AD- 1113 9525 1095-GGAGuuuAuucGGAAAAGcTsT 469 GCUUUUCCGAAuAAACUCCTsT 470 AD- 1113 96511096- GAGUUUAUUCGGAAAAGCCTsT 471 GGCUUUUCCGAAUAAACUCTsT 472 AD- 11149560 1096- GAGuuuAuucGGAAAAGccTsT 473 GGCUUUUCCGAAuAAACUCTsT 474 AD-1114 9686 1100- UUAUUCGGAAAAGCCAGCUTsT 475 AGCUGGCUUUUCCGAAUAATsT 476AD- 1118 9536 1100- uuAuucGGAAAAGccAGcuTsT 477 AGCUGGCUUUUCCGAAuAATsT478 AD- 1118 9662 1154- CCCUGGCGGGUGGGUACAGTsT 479CUGUACCCACCCGCCAGGGTsT 480 AD- 1172 9584 1154- cccuGGcGGGuGGGuAcAGTsT481 CUGuACCcACCCGCcAGGGTsT 482 AD- 1172 9710 1155- CCUGGCGGGUGGGUACAGCTT483 GCUGUACCCACCCGCCAGGTT 484 AD- 1173 15323 1157-UGGCGGGUGGGUACAGCCGTsT 485 CGGCUGUACCCACCCGCCATsT 486 AD- 1175 95511157- uGGcGGGuGGGuAcAGccGTsT 487 CGGCUGuACCcACCCGCcATsT 488 AD- 11759677 1158- GGCGGGUGGGUACAGCCGCTT 489 GCGGCUGUACCCACCCGCCTT 490 AD- 117615230 1162- GGUGGGUACAGCCGCGUCCTT 491 GGACGCGGCUGUACCCACCTT 492 AD- 118015231 1164- UGGGUACAGCCGCGUCCUCTT 493 GAGGACGCGGCUGUACCCATT 494 AD- 118215285 1172- GCCGCGUCCUCAACGCCGCTT 495 GCGGCGUUGAGGACGCGGCTT 496 AD- 119015396 1173- CCGCGUCCUCAACGCCGCCTT 497 GGCGGCGUUGAGGACGCGGTT 498 AD- 119115397 1216- GUCGUGCUGGUCACCGCUGTsT 499 CAGCGGUGACCAGCACGACTsT 500 AD-1234 9600 1216- GucGuGcuGGucAccGcuGTsT 501 cAGCGGUGACcAGcACGACTsT 502AD- 1234 9726 1217- UCGUGCUGGUCACCGCUGCTsT 503 GCAGCGGUGACCAGCACGATsT504 AD- 1235 9606 1217- ucGuGcuGGucAccGcuGcTsT 505GcAGCGGUGACcAGcACGATsT 506 AD- 1235 9732 1223- UGGUCACCGCUGCCGGCAATsT507 UUGCCGGCAGCGGUGACCATsT 508 AD- 1241 9633 1223-uGGucAccGcuGccGGcAATsT 509 UUGCCGGcAGCGGUGACcATsT 510 AD- 1241 97591224- GGUCACCGCUGCCGGCAACTsT 511 GUUGCCGGCAGCGGUGACCTsT 512 AD- 12429588 1224- GGucAccGcuGccGGcAAcTsT 513 GUUGCCGGcAGCGGUGACCTsT 514 AD-1242 9714 1227- CACCGCUGCCGGCAACUUCTsT 515 GAAGUUGCCGGCAGCGGUGTsT 516AD- 1245 9589 1227- cAccGcuGccGGcAAcuucTsT 517 GAAGUUGCCGGcAGCGGUGTsT518 AD- 1245 9715 1229 CCGCUGCCGGCAACUUCCGTsT 519 CGGAAGUUGCCGGCAGCGGTsT520 AD- 1247 9575 1229- ccGcuGccGGcAAcuuccGTsT 521CGGAAGUUGCCGGcAGCGGTsT 522 AD- 1247 9701 1230- CGCUGCCGGCAACUUCCGGTsT523 CCGGAAGUUGCCGGCAGCGTsT 524 AD- 1248 9563 1230-cGcuGccGGcAAcuuccGGTsT 525 CCGGAAGUUGCCGGcAGCGTsT 526 AD- 1248 96891231- GCUGCCGGCAACUUCCGGGTsT 527 CCCGGAAGUUGCCGGCAGCTsT 528 AD- 12499594 1231- GcuGccGGcAAcuuccGGGTsT 529 CCCGGAAGUUGCCGGcAGCTsT 530 AD-1249 9720 1236- CGGCAACUUCCGGGACGAUTsT 531 AUCGUCCCGGAAGUUGCCGTsT 532AD- 1254 9585 1236- cGGcAAcuuccGGGAcGAuTsT 533 AUCGUCCCGGAAGUUGCCGTsT534 AD- 1254 9711 1237- GGCAACUUCCGGGACGAUGTsT 535CAUCGUCCCGGAAGUUGCCTsT 536 AD- 1255 9614 1237- GGcAAcuuccGGGAcGAuGTsT537 cAUCGUCCCGGAAGUUGCCTsT 538 AD- 1255 9740 1243-UUCCGGGACGAUGCCUGCCTsT 539 GGCAGGCAUCGUCCCGGAATsT 540 AD- 1261 96151243- uuccGGGAcGAuGccuGccTsT 541 GGcAGGcAUCGUCCCGGAATsT 542 AD- 12619741 1248- GGACGAUGCCUGCCUCUACTsT 543 GUAGAGGCAGGCAUCGUCCTsT 544 AD-1266 9534 1248- GGACGAUGCCUGCCUCUACTsT 545 GUAGAGGCAGGCAUCGUCCTsT 546AD- 1266 9534 1248- GGAcGAuGccuGccucuAcTsT 547 GuAGAGGcAGGcAUCGUCCTsT548 AD- 1266 9660 1279 GCUCCCGAGGUCAUCACAGTT 549 CUGUGAUGACCUCGGGAGCTT550 AD- 1297 15324 1280- CUCCCGAGGUCAUCACAGUTT 551 ACUGUGAUGACCUCGGGAGTT552 AD- 1298 15232 1281- UCCCGAGGUCAUCACAGUUTT 553 AACUGUGAUGACCUCGGGATT554 AD- 1299 15233 1314- CCAAGACCAGCCGGUGACCTT 555 GGUCACCGGCUGGUCUUGGTT556 AD- 1332 15234 1315- CAAGACCAGCCGGUGACCCTT 557 GGGUCACCGGCUGGUCUUGTT558 AD- 1333 15286 1348- ACCAACUUUGGCCGCUGUGTsT 559CACAGCGGCCAAAGUUGGUTsT 560 AD- 1366 9590 1348- AccAAcuuuGGccGcuGuGTsT561 cAcAGCGGCcAAAGUUGGUTsT 562 AD- 1366 9716 1350-CAACUUUGGCCGCUGUGUGTsT 563 CACACAGCGGCCAAAGUUGTsT 564 AD- 1368 96321350- cAAcuuuGGccGcuGuGuGTsT 565 cAcAcAGCGGCcAAAGUUGTsT 566 AD- 13689758 1360- CGCUGUGUGGACCUCUUUGTsT 567 CAAAGAGGUCCACACAGCGTsT 568 AD-1378 9567 1360- cGcuGuGuGGAccucuuuGTsT 569 cAAAGAGGUCcAcAcAGCGTsT 570AD- 1378 9693 1390- GACAUCAUUGGUGCCUCCATsT 571 UGGAGGCACCAAUGAUGUCTsT572 AD- 1408 9586 1390- GAcAucAuuGGuGccuccATsT 573UGGAGGcACcAAUGAUGUCTsT 574 AD- 1408 9712 1394- UCAUUGGUGCCUCCAGCGATsT575 UCGCUGGAGGCACCAAUGATsT 576 AD- 1412 9564 1394-ucAuuGGuGccuccAGcGATsT 577 UCGCUGGAGGcACcAAUGATsT 578 AD- 1412 96901417- AGCACCUGCUUUGUGUCACTsT 579 GUGACACAAAGCAGGUGCUTsT 580 AD- 14359616 1417- AGcAccuGcuuuGuGucAcTsT 581 GUGAcAcAAAGcAGGUGCUTsT 582 AD-1435 9742 1433- CACAGAGUGGGACAUCACATT 583 UGUGAUGUCCCACUCUGUGTT 584 AD-1451 15398 1486- AUGCUGUCUGCCGAGCCGGTsT 585 CCGGCUCGGCAGACAGCAUTsT 586AD- 1504 9617 1486- AuGcuGucuGccGAGccGGTsT 587 CCGGCUCGGcAGAcAGcAUTsT588 AD- 1504 9743 1491- GUCUGCCGAGCCGGAGCUCTsT 589GAGCUCCGGCUCGGCAGACTsT 590 AD- 1509 9635 1491- GucuGccGAGccGGAGcucTsT591 GAGCUCCGGCUCGGcAGACTsT 592 AD- 1509 9761 1521-GUUGAGGCAGAGACUGAUCTsT 593 GAUCAGUCUCUGCCUCAACTsT 594 AD- 1539 95681521- GuuGAGGcAGAGAcuGAucTsT 595 GAUcAGUCUCUGCCUcAACTsT 596 AD- 15399694 1527- GCAGAGACUGAUCCACUUCTsT 597 GAAGUGGAUCAGUCUCUGCTsT 598 AD-1545 9576 1527- GcAGAGAcuGAuccAcuucTsT 599 GAAGUGGAUcAGUCUCUGCTsT 600AD- 1545 9702 1529- AGAGACUGAUCCACUUCUCTsT 601 GAGAAGUGGAUCAGUCUCUTsT602 AD- 1547 9627 1529- AGAGAcuGAuccAcuucucTsT 603GAGAAGUGGAUcAGUCUCUTsT 604 AD- 1547 9753 1543- UUCUCUGCCAAAGAUGUCATsT605 UGACAUCUUUGGCAGAGAATsT 606 AD- 1561 9628 1543-uucucuGccAAAGAuGucATsT 607 UGAcAUCUUUGGcAGAGAATsT 608 AD- 1561 97541545- CUCUGCCAAAGAUGUCAUCTsT 609 GAUGACAUCUUUGGCAGAGTsT 610 AD- 15639631 1545- cucuGccAAAGAuGucAucTsT 611 GAUGAcAUCUUUGGcAGAGTsT 612 AD-1563 9757 1580- CUGAGGACCAGCGGGUACUTsT 613 AGUACCCGCUGGUCCUCAGTsT 614AD- 1598 9595 1580- cuGAGGAccAGcGGGuAcuTsT 615 AGuACCCGCUGGUCCUcAGTsT616 AD- 1598 9721 1581- UGAGGACCAGCGGGUACUGTsT 617CAGUACCCGCUGGUCCUCATsT 618 AD- 1599 9544 1581- uGAGGAccAGcGGGuAcuGTsT619 cAGuACCCGCUGGUCCUcATsT 620 AD- 1599 9670 1666- ACUGUAUGGUCAGCACACUTT621 AGUGUGCUGACCAUACAGUTT 622 AD- 1684 15235 1668- UGUAUGGUCAGCACACUCGTT623 CGAGUGUGCUGACCAUACATT 624 AD- 1686 15236 1669- GUAUGGUCAGCACACUCGGTT625 CCGAGUGUGCUGACCAUACTT 626 AD- 1687 15168 1697 GGAUGGCCACAGCCGUCGCTT627 GCGACGGCUGUGGCCAUCCTT 628 AD- 1715 15174 1698 GAUGGCCACAGCCGUCGCCTT629 GGCGACGGCUGUGGCCAUCTT 630 AD- 1716 15325 1806 CAAGCUGGUCUGCCGGGCCTT631 GGCCCGGCAGACCAGCUUGTT 632 AD- 1824 15326 1815-CUGCCGGGCCCACAACGCUTsT 633 AGCGUUGUGGGCCCGGCAGTsT 634 AD- 1833 95701815- cuGccGGGcccAcAAcGcuTsT 635 AGCGUUGUGGGCCCGGcAGTsT 636 AD- 18339696 1816- UGCCGGGCCCACAACGCUUTsT 637 AAGCGUUGUGGGCCCGGCATsT 638 AD-1834 9566 1816- uGccGGGcccAcAAcGcuuTsT 639 AAGCGUUGUGGGCCCGGcATsT 640AD- 1834 9692 1818- CCGGGCCCACAACGCUUUUTsT 641 AAAAGCGUUGUGGGCCCGGTsT642 AD- 1836 9532 1818- ccGGGcccAcAAcGcuuuuTsT 643AAAAGCGUUGUGGGCCCGGTsT 644 AD- 1836 9658 1820- GGGCCCACAACGCUUUUGGTsT645 CCAAAAGCGUUGUGGGCCCTsT 646 AD- 1838 9549 1820-GGGcccAcAAcGcuuuuGGTsT 647 CcAAAAGCGUUGUGGGCCCTsT 648 AD- 1838 96751840- GGUGAGGGUGUCUACGCCATsT 649 UGGCGUAGACACCCUCACCTsT 650 AD- 18589541 1840- GGuGAGGGuGucuAcGccATsT 651 UGGCGuAGAcACCCUcACCTsT 652 AD-1858 9667 1843- GAGGGUGUCUACGCCAUUGTsT 653 CAAUGGCGUAGACACCCUCTsT 654AD- 1861 9550 1843- GAGGGuGucuAcGccAuuGTsT 655 cAAUGGCGuAGAcACCCUCTsT656 AD- 1861 9676 1861- GCCAGGUGCUGCCUGCUACTsT 657GUAGCAGGCAGCACCUGGCTsT 658 AD- 1879 9571 1861- GccAGGuGcuGccuGcuAcTsT659 GuAGcAGGcAGcACCUGGCTsT 660 AD- 1879 9697 1862-CCAGGUGCUGCCUGCUACCTsT 661 GGUAGCAGGCAGCACCUGGTsT 662 AD- 1880 95721862- ccAGGuGcuGccuGcuAccTsT 663 GGuAGcAGGcAGcACCUGGTsT 664 AD- 18809698 2008- ACCCACAAGCCGCCUGUGCTT 665 GCACAGGCGGCUUGUGGGUTT 666 AD- 202615327 2023- GUGCUGAGGCCACGAGGUCTsT 667 GACCUCGUGGCCUCAGCACTsT 668 AD-2041 9639 2023- GuGcuGAGGccAcGAGGucTsT 669 GACCUCGUGGCCUcAGcACTsT 670AD- 2041 9765 2024- UGCUGAGGCCACGAGGUCATsT 671 UGACCUCGUGGCCUCAGCATsT672 AD- 2042 9518 2024- UGCUGAGGCCACGAGGUCATsT 673UGACCUCGUGGCCUCAGCATsT 674 AD- 2042 9518 2024- uGcuGAGGccAcGAGGucATsT675 UGACCUCGUGGCCUcAGcATsT 676 AD- 2042 9644 2024-UfgCfuGfaGfgCfcAfcGfaGfgUfcA 677 p- 678 AD- 2042 fTsTuGfaCfcUfcGfuGfgCfcUfcAfgCfaTsT 14672 2024- UfGCfUfGAGGCfCfACfGAGGUfCfAT679 UfGACfCfUfCfGUfGGCfCfUfCfAGCfAT 680 AD- 2042 sT sT 14682 2024-UgCuGaGgCcAcGaGgUcATsT 681 p- 682 AD- 2042uGfaCfcUfcGfuGfgCfcUfcAfgCfaTsT 14692 2024- UgCuGaGgCcAcGaGgUcATsT 683UfGACfCfUfCfGUfGGCfCfUfCfAGCfAT 684 AD- 2042 sT 14702 2024-UfgCfuGfaGfgCfcAfcGfaGfgUfcA 685 UGACCucGUggCCUCAgcaTsT 686 AD- 2042fTsT 14712 2024- UfGCfUfGAGGCfCfACfGAGGUfCfAT 687 UGACCucGUggCCUCAgcaTsT688 AD- 2042 sT 14722 2024- UgCuGaGgCcAcGaGgUcATsT 689UGACCucGUggCCUCAgcaTsT 690 AD- 2042 14732 2024-GfuGfgUfcAfgCfgGfcCfgGfgAfuG 691 p- 692 AD- 2042 fTsTcAfuCfcCfgGfcCfgCfuGfaCfcAfcTsT 15078 2024- GUfGGUfCfAGCfGGCfCfGGGAUfGTs693 CfAUfCfCfCfGGCfCfGCfUfGACfCfACf 694 AD- 2042 T TsT 15088 2024-GuGgUcAgCgGcCgGgAuGTsT 695 p- 696 AD- 2042cAfuCfcCfgGfcCfgCfuGfaCfcAfcTsT 15098 2024- GuGgUcAgCgGcCgGgAuGTsT 697CfAUfCfCfCfGGCfCfGCfUfGACfCfACf 698 AD- 2042 TsT 15108 2024-GfuGfgUfcAfgCfgGfcCfgGfgAfuG 699 CAUCCcgGCcgCUGACcacTsT 700 AD- 2042fTsT 15118 2024- GUfGGUfCfAGCfGGCfCfGGGAUfGTs 701 CAUCCcgGCcgCUGACcacTsT702 AD- 2042 T 15128 2024- GuGgUcAgCgGcCgGgAuGTsT 703CAUCCcgGCcgCUGACcacTsT 704 AD- 2042 15138 2030- GGCCACGAGGUCAGCCCAATT705 UUGGGCUGACCUCGUGGCCTT 706 AD- 2048 15237 2035- CGAGGUCAGCCCAACCAGUTT707 ACUGGUUGGGCUGACCUCGTT 708 AD- 2053 15287 2039- GUCAGCCCAACCAGUGCGUTT709 ACGCACUGGUUGGGCUGACTT 710 AD- 2057 15238 2041- CAGCCCAACCAGUGCGUGGTT711 CCACGCACUGGUUGGGCUGTT 712 AD- 2059 15328 2062- CACAGGGAGGCCAGCAUCCTT713 GGAUGCUGGCCUCCCUGUGTT 714 AD- 2080 15399 2072-CCAGCAUCCACGCUUCCUGTsT 715 CAGGAAGCGUGGAUGCUGGTsT 716 A D- 2090 95822072- ccAGcAuccAcGcuuccuGTsT 717 cAGGAAGCGUGGAUGCUGGTsT 718 AD- 20909708 2118- AGUCAAGGAGCAUGGAAUCTsT 719 GAUUCCAUGCUCCUUGACUTsT 720 AD-2136 9545 2118- AGucAAGGAGcAuGGAAucTsT 721 GAUUCcAUGCUCCUUGACUTsT 722AD- 2136 9671 2118- AfgUfcAfaGfgAfgCfaUfgGfaAfuC 723 p- 724 AD- 2136fTsT gAfuUfcCfaUfgCfuCfcUfuGfaCfuTsT 14674 2118-AGUfCfAAGGAGCfAUfGGAAUfCfTsT 725 GAUfUfCfCfAUfGCfUfCfCfUfUfGACfU 726 AD-2136 fTsT 14684 2118- AgUcAaGgAgCaUgGaAuCTsT 727 p- 728 AD- 2136gAfuUfcCfaUfgCfuCfcUfuGfaCfuTsT 14694 2118- AgUcAaGgAgCaUgGaAuCTsT 729GAUfUfCfCfAUfGCfUfCfCfUfUfGACfU 730 AD- 2136 fTsT 14704 2118-AfgUfcAfaGfgAfgCfaUfgGfaAfuC 731 GAUUCcaUGcuCCUUGacuTsT 732 AD- 2136fTsT 14714 2118- AGUfCfAAGGAGCfAUfGGAAUfCfTsT 733 GAUUCcaUGcuCCUUGacuTsT734 AD- 2136 14724 2118- AgUcAaGgAgCaUgGaAuCTsT 735GAUUCcaUGcuCCUUGacuTsT 736 AD- 2136 14734 2118-GfcGfgCfaCfcCfuCfaUfaGfgCfcU 737 p- 738 AD- 2136 fTsTaGfgCfcUfaUfgAfgGfgUfgCfcGfcTsT 15080 2118- GCfGGCfACfCfCfUfCfAUfAGGCfCf739 AGGCfCfUfAUfGAGGGUfGCfCfGCfTsT 740 AD- 2136 UfTsT 15090 2118-GcGgCaCcCuCaUaGgCcUTsT 741 P- 742 AD- 2136aGfgCfcUfaUfgAfgGfgUfgCfcGfcTsT 15100 2118- GcGgCaCcCuCaUaGgCcUTsT 743AGGCfCfUfAUfGAGGGUfGCfCfGCfTsT 744 AD- 2136 15110 2118-GfcGfgCfaCfcCfuCfaUfaGfgCfcU 745 AGGCCuaUGagGGUGCcgcTsT 746 AD- 2136fTsT 15120 2118- GCfGGCfACfCfCfUfCfAUfAGGCfCf 747 AGGCCuaUGagGGUGCcgcTsT748 AD- 2136 UfTsT 15130 2118- GcGgCaCcCuCaUaGgCcUTsT 749AGGCCuaUGagGGUGCcgcTsT 750 AD- 2136 15140 2122- AAGGAGCAUGGAAUCCCGGTsT751 CCGGGAUUCCAUGCUCCUUTsT 752 AD- 2140 9522 2122-AAGGAGcAuGGAAucccGGTsT 753 CCGGGAUUCcAUGCUCCUUTsT 754 AD- 2140 96482123- AGGAGCAUGGAAUCCCGGCTsT 755 GCCGGGAUUCCAUGCUCCUTsT 756 AD- 21419552 2123- AGGAGcAuGGAAucccGGcTsT 757 GCCGGGAUUCcAUGCUCCUTsT 758 AD-2141 9678 2125- GAGCAUGGAAUCCCGGCCCTsT 759 GGGCCGGGAUUCCAUGCUCTsT 760AD- 2143 9618 2125- GAGcAuGGAAucccGGcccTsT 761 GGGCCGGGAUUCcAUGCUCTsT762 AD- 2143 9744 2230- GCCUACGCCGUAGACAACATT 763 UGUUGUCUACGGCGUAGGCTT764 AD- 2248 15239 2231- CCUACGCCGUAGACAACACTT 765 GUGUUGUCUACGGCGUAGGTT766 AD- 2249 15212 2232- CUACGCCGUAGACAACACGTT 767 CGUGUUGUCUACGGCGUAGTT768 AD- 2250 15240 2233- UACGCCGUAGACAACACGUTT 769 ACGUGUUGUCUACGGCGUATT770 AD- 2251 15177 2235- CGCCGUAGACAACACGUGUTT 771 ACACGUGUUGUCUACGGCGTT772 AD- 2253 15179 2236- GCCGUAGACAACACGUGUGTT 773 CACACGUGUUGUCUACGGCTT774 AD- 2254 15180 2237- CCGUAGACAACACGUGUGUTT 775 ACACACGUGUUGUCUACGGTT776 AD- 2255 15241 2238- CGUAGACAACACGUGUGUATT 777 UACACACGUGUUGUCUACGTT778 AD- 2256 15268 2240- UAGACAACACGUGUGUAGUTT 779 ACUACACACGUGUUGUCUATT780 AD- 2258 15242 2241- AGACAACACGUGUGUAGUCTT 781 GACUACACACGUGUUGUCUTT782 AD- 2259 15216 2242- GACAACACGUGUGUAGUCATT 783 UGACUACACACGUGUUGUCTT784 AD- 2260 15176 2243- ACAACACGUGUGUAGUCAGTT 785 CUGACUACACACGUGUUGUTT786 AD- 2261 15181 2244 CAACACGUGUGUAGUCAGGTT 787 CCUGACUACACACGUGUUGTT788 AD- 2262 15243 2247- CACGUGUGUAGUCAGGAGCTT 789 GCUCCUGACUACACACGUGTT790 AD- 2265 15182 2248- ACGUGUGUAGUCAGGAGCCTT 791 GGCUCCUGACUACACACGUTT792 AD- 2266 15244 2249 CGUGUGUAGUCAGGAGCCGTT 793 CGGCUCCUGACUACACACGTT794 AD- 2267 15387 2251- UGUGUAGUCAGGAGCCGGGTT 795 CCCGGCUCCUGACUACACATT796 AD- 2269 15245 2257- GUCAGGAGCCGGGACGUCATsT 797UGACGUCCCGGCUCCUGACTsT 798 AD- 2275 9555 2257- GucAGGAGccGGGAcGucATsT799 UGACGUCCCGGCUCCUGACTsT 800 AD- 2275 9681 2258-UCAGGAGCCGGGACGUCAGTsT 801 CUGACGUCCCGGCUCCUGATsT 802 AD- 2276 96192258- ucAGGAGccGGGAcGucAGTsT 803 CUGACGUCCCGGCUCCUGATsT 804 AD- 22769745 2259- CAGGAGCCGGGACGUCAGCTsT 805 GCUGACGUCCCGGCUCCUGTsT 806 AD-2277 9620 2259- cAGGAGccGGGAcGucAGcTsT 807 GCUGACGUCCCGGCUCCUGTsT 808AD- 2277 9746 2263- AGCCGGGACGUCAGCACUATT 809 UAGUGCUGACGUCCCGGCUTT 810AD- 2281 15288 2265- CCGGGACGUCAGCACUACATT 811 UGUAGUGCUGACGUCCCGGTT 812AD- 2283 15246 2303- CCGUGACAGCCGUUGCCAUTT 813 AUGGCAACGGCUGUCACGGTT 814AD- 2321 15289 2317- GCCAUCUGCUGCCGGAGCCTsT 815 GGCUCCGGCAGCAGAUGGCTsT816 AD- 2335 9324 2375- CCCAUCCCAGGAUGGGUGUTT 817 ACACCCAUCCUGGGAUGGGTT818 AD- 2393 15329 2377- CAUCCCAGGAUGGGUGUCUTT 819 AGACACCCAUCCUGGGAUGTT820 AD- 2395 15330 2420- AGCUUUAAAAUGGUUCCGATT 821 UCGGAACCAUUUUAAAGCUTT822 AD- 2438 15169 2421- GCUUUAAAAUGGUUCCGACTT 823 GUCGGAACCAUUUUAAAGCTT824 AD- 2439 15201 2422- CUUUAAAAUGGUUCCGACUTT 825 AGUCGGAACCAUUUUAAAGTT826 AD- 2440 15331 2423- UUUAAAAUGGUUCCGACUUTT 827 AAGUCGGAACCAUUUUAAATT828 AD- 2441 15190 2424- UUAAAAUGGUUCCGACUUGTT 829 CAAGUCGGAACCAUUUUAATT830 AD- 2442 15247 2425- UAAAAUGGUUCCGACUUGUTT 831 ACAAGUCGGAACCAUUUUATT832 AD- 2443 15248 2426- AAAAUGGUUCCGACUUGUCTT 833 GACAAGUCGGAACCAUUUUTT834 AD- 2444 15175 2427- AAAUGGUUCCGACUUGUCCTT 835 GGACAAGUCGGAACCAUUUTT836 AD- 2445 15249 2428- AAUGGUUCCGACUUGUCCCTT 837 GGGACAAGUCGGAACCAUUTT838 AD- 2446 15250 2431- GGUUCCGACUUGUCCCUCUTT 839 AGAGGGACAAGUCGGAACCTT840 AD- 2449 15400 2457- CUCCAUGGCCUGGCACGAGTT 841 CUCGUGCCAGGCCAUGGAGTT842 AD- 2475 15332 2459- CCAUGGCCUGGCACGAGGGTT 843 CCCUCGUGCCAGGCCAUGGTT844 AD- 2477 15388 2545- GAACUCACUCACUCUGGGUTT 845 ACCCAGAGUGAGUGAGUUCTT846 AD- 2563 15333 2549- UCACUCACUCUGGGUGCCUTT 847 AGGCACCCAGAGUGAGUGATT848 AD- 2567 15334 2616- UUUCACCAUUCAAACAGGUTT 849 ACCUGUUUGAAUGGUGAAATT850 AD- 2634 15335 2622- CAUUCAAACAGGUCGAGCUTT 851 AGCUCGACCUGUUUGAAUGTT852 AD- 2640 15183 2623- AUUCAAACAGGUCGAGCUGTT 853 CAGCUCGACCUGUUUGAAUTT854 AD- 2641 15202 2624- UUCAAACAGGUCGAGCUGUTT 855 ACAGCUCGACCUGUUUGAATT856 AD- 2642 15203 2625- UCAAACAGGUCGAGCUGUGTT 857 CACAGCUCGACCUGUUUGATT858 AD- 2643 15272 2626- CAAACAGGUCGAGCUGUGCTT 859 GCACAGCUCGACCUGUUUGTT860 AD- 2644 15217 2627- AAACAGGUCGAGCUGUGCUTT 861 AGCACAGCUCGACCUGUUUTT862 AD- 2645 15290 2628- AACAGGUCGAGCUGUGCUCTT 863 GAGCACAGCUCGACCUGUUTT864 AD- 2646 15218 2630- CAGGUCGAGCUGUGCUCGGTT 865 CCGAGCACAGCUCGACCUGTT866 AD- 2648 15389 2631- AGGUCGAGCUGUGCUCGGGTT 867 CCCGAGCACAGCUCGACCUTT868 AD- 2649 15336 2633- GUCGAGCUGUGCUCGGGUGTT 869 CACCCGAGCACAGCUCGACTT870 AD- 2651 15337 2634- UCGAGCUGUGCUCGGGUGCTT 871 GCACCCGAGCACAGCUCGATT872 AD- 2652 15191 2657- AGCUGCUCCCAAUGUGCCGTT 873 CGGCACAUUGGGAGCAGCUTT874 AD- 2675 15390 2658- GCUGCUCCCAAUGUGCCGATT 875 UCGGCACAUUGGGAGCAGCTT876 AD- 2676 15338 2660- UGCUCCCAAUGUGCCGAUGTT 877 CAUCGGCACAUUGGGAGCATT878 AD- 2678 15204 2663- UCCCAAUGUGCCGAUGUCCTT 879 GGACAUCGGCACAUUGGGATT880 AD- 2681 15251 2665- CCAAUGUGCCGAUGUCCGUTT 881 ACGGACAUCGGCACAUUGGTT882 AD- 2683 15205 2666- CAAUGUGCCGAUGUCCGUGTT 883 CACGGACAUCGGCACAUUGTT884 AD- 2684 15171 2667- AAUGUGCCGAUGUCCGUGGTT 885 CCACGGACAUCGGCACAUUTT886 AD- 2685 15252 2673- CCGAUGUCCGUGGGCAGAATT 887 UUCUGCCCACGGACAUCGGTT888 AD- 2691 15339 2675- GAUGUCCGUGGGCAGAAUGTT 889 CAUUCUGCCCACGGACAUCTT890 AD- 2693 15253 2678- GUCCGUGGGCAGAAUGACUTT 891 AGUCAUUCUGCCCACGGACTT892 AD- 2696 15340 2679- UCCGUGGGCAGAAUGACUUTT 893 AAGUCAUUCUGCCCACGGATT894 AD- 2697 15291 2683- UGGGCAGAAUGACUUUUAUTT 895 AUAAAAGUCAUUCUGCCCATT896 AD- 2701 15341 2694- ACUUUUAUUGAGCUCUUGUTT 897 ACAAGAGCUCAAUAAAAGUTT898 AD- 2712 15401 2700- AUUGAGCUCUUGUUCCGUGTT 899 CACGGAACAAGAGCUCAAUTT900 AD- 2718 15342 2704- AGCUCUUGUUCCGUGCCAGTT 901 CUGGCACGGAACAAGAGCUTT902 AD- 2722 15343 2705- GCUCUUGUUCCGUGCCAGGTT 903 CCUGGCACGGAACAAGAGCTT904 AD- 2723 15292 2710- UGUUCCGUGCCAGGCAUUCTT 905 GAAUGCCUGGCACGGAACATT906 AD- 2728 15344 2711- GUUCCGUGCCAGGCAUUCATT 907 UGAAUGCCUGGCACGGAACTT908 AD- 2729 15254 2712- UUCCGUGCCAGGCAUUCAATT 909 UUGAAUGCCUGGCACGGAATT910 AD- 2730 15345 2715- CGUGCCAGGCAUUCAAUCCTT 911 GGAUUGAAUGCCUGGCACGTT912 AD- 2733 15206 2716- GUGCCAGGCAUUCAAUCCUTT 913 AGGAUUGAAUGCCUGGCACTT914 AD- 2734 15346 2728- CAAUCCUCAGGUCUCCACCTT 915 GGUGGAGACCUGAGGAUUGTT916 AD- 2746 15347 2743- CACCAAGGAGGCAGGAUUCTsT 917GAAUCCUGCCUCCUUGGUGTsT 918 AD- 2761 9577 2743- cAccAAGGAGGcAGGAuucTsT919 GAAUCCUGCCUCCUUGGUGTsT 920 AD- 2761 9703 2743-CfaCfcAfaGfgAfgGfcAfgGfaUfuC 921 p- 922 AD- 2761 fTsTgAfaUfcCfuGfcCfuCfcUfuGfgUfgTsT 14678 2743- CfACfCfAAGGAGGCfAGGAUfUfCfTs923 GAAUfCfCfUfGCfCfUfCfCfUfUfGGUfG 924 AD- 2761 T TsT 14688 2743-CaCcAaGgAgGcAgGaUuCTsT 925 p- 926 AD- 2761gAfaUfcCfuGfcCfuCfcUfuGfgUfgTsT 14698 2743- CaCcAaGgAgGcAgGaUuCTsT 927GAAUfCfCfUfGCfCfUfCfCfUfUfGGUfG 928 AD- 2761 TsT 14708 2743-CfaCfcAfaGfgAfgGfcAfgGfaUfuC 929 GAAUCcuGCcuCCUUGgugTsT 930 AD- 2761fTsT 14718 2743- CfACfCfAAGGAGGCfAGGAUfUfCfTs 931 GAAUCcuGCcuCCUUGgugTsT932 AD- 2761 T 14728 2743- CaCcAaGgAgGcAgGaUuCTsT 933GAAUCcuGCcuCCUUGgugTsT 934 AD- 2761 14738 2743-GfgCfcUfgGfaGfuUfuAfuUfcGfgA 935 p- 936 AD- 2761 fTsTuCfcGfaAfuAfaAfcUfcCfaGfgCfcTsT 15084 2743- GGCfCfUfGGAGUfUfUfAUfUfCfGGA937 UfCfCfGAAUfAAACfUfCfCfAGGCfCfTs 938 AD- 2761 TsT T 15094 2743-GgCcUgGaGuUuAuUcGgATsT 939 p- 940 AD- 2761uCfcGfaAfuAfaAfcUfcCfaGfgCfcTsT 15104 2743- GgCcUgGaGuUuAuUcGgATsT 941UfCfCfGAAUfAAACfUfCfCfAGGCfCfTs 942 AD- 2761 T 15114 2743-GfgCfcUfgGfaGfuUfuAfuUfcGfgA 943 UCCGAauAAacUCCAGgccTsT 944 AD- 2761fTsT 15124 2743- GGCfCfUfGGAGUfUfUfAUfUfCfGGA 945 UCCGAauAAacUCCAGgccTsT946 AD- 2761 TsT 15134 2743- Gg CcUgGaGuUuAuUcGgATsT 947UCCGAauAAacUCCAGgccTsT 948 AD- 2761 15144 2753- GCAGGAUUCUUCCCAUGGATT949 UCCAUGGGAAGAAUCCUGCTT 950 AD- 2771 15391 2794- UGCAGGGACAAACAUCGUUTT951 AACGAUGUUUGUCCCUGCATT 952 AD- 2812 15348 2795- GCAGGGACAAACAUCGUUGTT953 CAACGAUGUUUGUCCCUGCTT 954 AD- 2813 15349 2797- AGGGACAAACAUCGUUGGGTT955 CCCAACGAUGUUUGUCCCUTT 956 AD- 2815 15170 2841- CCCUCAUCUCCAGCUAACUTT957 AGUUAGCUGGAGAUGAGGGTT 958 AD- 2859 15350 2845- CAUCUCCAGCUAACUGUGGTT959 CCACAGUUAGCUGGAGAUGTT 960 AD- 2863 15402 2878- GCUCCCUGAUUAAUGGAGGTT961 CCUCCAUUAAUCAGGGAGCTT 962 AD- 2896 15293 2881- CCCUGAUUAAUGGAGGCUUTT963 AAGCCUCCAUUAAUCAGGGTT 964 AD- 2899 15351 2882- CCUGAUUAAUGGAGGCUUATT965 UAAGCCUCCAUUAAUCAGGTT 966 AD- 2900 15403 2884- UGAUUAAUGGAGGCUUAGCTT967 GCUAAGCCUCCAUUAAUCATT 968 AD- 2902 15404 2885- GAUUAAUGGAGGCUUAGCUTT969 AGCUAAGCCUCCAUUAAUCTT 970 AD- 2903 15207 2886- AUUAAUGGAGGCUUAGCUUTT971 AAGCUAAGCCUCCAUUAAUTT 972 AD- 2904 15352 2887- UUAAUGGAGGCUUAGCUUUTT973 AAAGCUAAGCCUCCAUUAATT 974 AD- 2905 15255 2903-UUUCUGGAUGGCAUCUAGCTsT 975 GCUAGAUGCCAUCCAGAAATsT 976 AD- 2921 96032903- uuucuGGAuGGcAucuAGcTsT 977 GCuAGAUGCcAUCcAGAAATsT 978 AD- 29219729 2904- UUCUGGAUGGCAUCUAGCCTsT 979 GGCUAGAUGCCAUCCAGAATsT 980 AD-2922 9599 2904- uucuGGAuGGcAucuAGccTsT 981 GGCuAGAUGCcAUCcAGAATsT 982AD- 2922 9725 2905- UCUGGAUGGCAUCUAGCCATsT 983 UGGCUAGAUGCCAUCCAGATsT984 AD- 2923 9621 2905- ucuGGAuGGcAucuAGccATsT 985UGGCuAGAUGCcAUCcAGATsT 986 AD- 2923 9747 2925- AGGCUGGAGACAGGUGCGCTT 987GCGCACCUGUCUCCAGCCUTT 988 AD- 2943 15405 2926- GGCUGGAGACAGGUGCGCCTT 989GGCGCACCUGUCUCCAGCCTT 990 AD- 2944 15353 2927- GCUGGAGACAGGUGCGCCCTT 991GGGCGCACCUGUCUCCAGCTT 992 AD- 2945 15354 2972- UUCCUGAGCCACCUUUACUTT 993AGUAAAGGUGGCUCAGGAATT 994 AD- 2990 15406 2973- UCCUGAGCCACCUUUACUCTT 995GAGUAAAGGUGGCUCAGGATT 996 AD- 2991 15407 2974- CCUGAGCCACCUUUACUCUTT 997AGAGUAAAGGUGGCUCAGGTT 998 AD- 2992 15355 2976- UGAGCCACCUUUACUCUGCTT 999GCAGAGUAAAGGUGGCUCATT 1000 AD- 2994 15356 2978- AGCCACCUUUACUCUGCUCTT1001 GAGCAGAGUAAAGGUGGCUTT 1002 AD- 2996 15357 2981-CACCUUUACUCUGCUCUAUTT 1003 AUAGAGCAGAGUAAAGGUGTT 1004 AD- 2999 152692987- UACUCUGCUCUAUGCCAGGTsT 1005 CCUGGCAUAGAGCAGAGUATsT 1006 AD- 30059565 2987- uAcucuGcucuAuGccAGGTsT 1007 CCUGGcAuAGAGcAGAGuATsT 1008 AD-3005 9691 2998- AUGCCAGGCUGUGCUAGCATT 1009 UGCUAGCACAGCCUGGCAUTT 1010AD- 3016 15358 3003- AGGCUGUGCUAGCAACACCTT 1011 GGUGUUGCUAGCACAGCCUTT1012 AD- 3021 15359 3006- CUGUGCUAGCAACACCCAATT 1013UUGGGUGUUGCUAGCACAGTT 1014 AD- 3024 15360 3010- GCUAGCAACACCCAAAGGUTT1015 ACCUUUGGGUGUUGCUAGCTT 1016 AD- 3028 15219 3038-GGAGCCAUCACCUAGGACUTT 1017 AGUCCUAGGUGAUGGCUCCTT 1018 AD- 3056 153613046- CACCUAGGACUGACUCGGCTT 1019 GCCGAGUCAGUCCUAGGUGTT 1020 AD- 306415273 3051- AGGACUGACUCGGCAGUGUTT 1021 ACACUGCCGAGUCAGUCCUTT 1022 AD-3069 15362 3052- GGACUGACUCGGCAGUGUGTT 1023 CACACUGCCGAGUCAGUCCTT 1024AD- 3070 15192 3074- UGGUGCAUGCACUGUCUCATT 1025 UGAGACAGUGCAUGCACCATT1026 AD- 3092 15256 3080- AUGCACUGUCUCAGCCAACTT 1027GUUGGCUGAGACAGUGCAUTT 1028 AD- 3098 15363 3085- CUGUCUCAGCCAACCCGCUTT1029 AGCGGGUUGGCUGAGACAGTT 1030 AD- 3103 15364 3089-CUCAGCCAACCCGCUCCACTsT 1031 GUGGAGCGGGUUGGCUGAGTsT 1032 AD- 3107 96043089- cucAGccAAcccGcuccAcTsT 1033 GUGGAGCGGGUUGGCUGAGTsT 1034 AD- 31079730 3093- GCCAACCCGCUCCACUACCTsT 1035 GGUAGUGGAGCGGGUUGGCTsT 1036 AD-3111 9527 3093- GccAAcccGcuccAcuAccTsT 1037 GGuAGUGGAGCGGGUUGGCTsT 1038AD- 3111 9653 3096- AACCCGCUCCACUACCCGGTT 1039 CCGGGUAGUGGAGCGGGUUTT1040 AD- 3114 15365 3099- CCGCUCCACUACCCGGCAGTT 1041CUGCCGGGUAGUGGAGCGGTT 1042 AD- 3117 15294 3107- CUACCCGGCAGGGUACACATT1043 UGUGUACCCUGCCGGGUAGTT 1044 AD- 3125 15173 3108-UACCCGGCAGGGUACACAUTT 1045 AUGUGUACCCUGCCGGGUATT 1046 AD- 3126 153663109- ACCCGGCAGGGUACACAUUTT 1047 AAUGUGUACCCUGCCGGGUTT 1048 AD- 312715367 3110- CCCGGCAGGGUACACAUUCTT 1049 GAAUGUGUACCCUGCCGGGTT 1050 AD-3128 15257 3112- CGGCAGGGUACACAUUCGCTT 1051 GCGAAUGUGUACCCUGCCGTT 1052AD- 3130 15184 3114- GCAGGGUACACAUUCGCACTT 1053 GUGCGAAUGUGUACCCUGCTT1054 AD- 3132 15185 3115- CAGGGUACACAUUCGCACCTT 1055GGUGCGAAUGUGUACCCUGTT 1056 AD- 3133 15258 3116- AGGGUACACAUUCGCACCCTT1057 GGGUGCGAAUGUGUACCCUTT 1058 AD- 3134 15186 3196-GGAACUGAGCCAGAAACGCTT 1059 GCGUUUCUGGCUCAGUUCCTT 1060 AD- 3214 152743197- GAACUGAGCCAGAAACGCATT 1061 UGCGUUUCUGGCUCAGUUCTT 1062 AD- 321515368 3198- AACUGAGCCAGAAACGCAGTT 1063 CUGCGUUUCUGGCUCAGUUTT 1064 AD-3216 15369 3201- UGAGCCAGAAACGCAGAUUTT 1065 AAUCUGCGUUUCUGGCUCATT 1066AD- 3219 15370 3207- AGAAACGCAGAUUGGGCUGTT 1067 CAGCCCAAUCUGCGUUUCUTT1068 AD- 3225 15259 3210- AACGCAGAUUGGGCUGGCUTT 1069AGCCAGCCCAAUCUGCGUUTT 1070 AD- 3228 15408 3233- AGCCAAGCCUCUUCUUACUTsT1071 AGUAAGAAGAGGCUUGGCUTsT 1072 AD- 3251 9597 3233-AGccAAGccucuucuuAcuTsT 1073 AGuAAGAAGAGGCUUGGCUTsT 1074 AD- 3251 97233233- AfgCfcAfaGfcCfuCfuUfcUfuAfcU 1075 p- 1076 AD- 3251 fTsTaGfuAfaGfaAfgAfgGfcUfuGfgCfuTsT 14680 3233- AGCfCfAAGCfCfUfCfUfUfCfUfUfA1077 AGUfAAGAAGAGGCfUfUfGGCfUfTsT 1078 AD- 3251 CfUfTsT 14690 3233-AgCcAaGcCuCuUcUuAcUTsT 1079 p- 1080 AD- 3251aGfuAfaGfaAfgAfgGfcUfuGfgCfuTsT 14700 3233- AgCcAaGcCuCuUcUuAcUTsT 1081AGUfAAGAAGAGGCfUfUfGGCfUfTsT 1082 AD- 3251 14710 3233-AfgCfcAfaGfcCfuCfuUfcUfuAfcU 1083 AGUAAgaAGagGCUUGgcuTsT 1084 AD- 3251fTsT 14720 3233- AGCfCfAAGCfCfUfCfUfUfCfUfUfA 1085AGUAAgaAGagGCUUGgcuTsT 1086 AD- 3251 CfUfTsT 14730 3233-AgCcAaGcCuCuUcUuAcUTsT 1087 AGUAAgaAGagGCUUGgcuTsT 1088 AD- 3251 147403233- UfgGfuUfcCfcUfgAfgGfaCfcAfgC 1089 p- 1090 AD- 3251 fTsTgCfuGfgUfcCfuCfaGfgGfaAfcCfaTsT 15086 3233- UfGGUfUfCfCfCfUfGAGGACfCfAGC1091 GCfUfGGUfCfCfUfCfAGGGAACfCfATsT 1092 AD- 3251 fTsT 15096 3233-UgGuUcCcUgAgGaCcAgCTsT 1093 p- 1094 AD- 3251gCfuGfgUfcCfuCfaGfgGfaAfcCfaTsT 15106 3233- UgGuUcCcUgAgGaCcAgCTsT 1095GCfUfGGUfCfCfUfCfAGGGAACfCfATsT 1096 AD- 3251 15116 3233-UfgGfuUfcCfcUfgAfgGfaCfcAfgC 1097 GCUGGucCUcaGGGAAccaTsT 1098 AD- 3251fTsT 15126 3233- UfGGUfUfCfCfCfUfGAGGACfCfAGC 1099GCUGGucCUcaGGGAAccaTsT 1100 AD- 3251 fTsT 15136 3233-UgGuUcCcUgAgGaCcAgCTsT 1101 GCUGGucCUcaGGGAAccaTsT 1102 AD- 3251 151463242- UCUUCUUACUUCACCCGGCTT 1103 GCCGGGUGAAGUAAGAAGATT 1104 AD- 326015260 3243- CUUCUUACUUCACCCGGCUTT 1105 AGCCGGGUGAAGUAAGAAGTT 1106 AD-3261 15371 3244- UUCUUACUUCACCCGGCUGTT 1107 CAGCCGGGUGAAGUAAGAATT 1108AD- 3262 15372 3262- GGGCUCCUCAUUUUUACGGTT 1109 CCGUAAAAAUGAGGAGCCCTT1110 AD- 3280 15172 3263- GGCUCCUCAUUUUUACGGGTT 1111CCCGUAAAAAUGAGGAGCCTT 1112 AD- 3281 15295 3264- GCUCCUCAUUUUUACGGGUTT1113 ACCCGUAAAAAUGAGGAGCTT 1114 AD- 3282 15373 3265-CUCCUCAUUUUUACGGGUATT 1115 UACCCGUAAAAAUGAGGAGTT 1116 AD- 3283 151633266- UCCUCAUUUUUACGGGUAATT 1117 UUACCCGUAAAAAUGAGGATT 1118 AD- 328415165 3267- CCUCAUUUUUACGGGUAACTT 1119 GUUACCCGUAAAAAUGAGGTT 1120 AD-3285 15374 3268- CUCAUUUUUACGGGUAACATT 1121 UGUUACCCGUAAAAAUGAGTT 1122AD- 3286 15296 3270- CAUUUUUACGGGUAACAGUTT 1123 ACUGUUACCCGUAAAAAUGTT1124 AD- 3288 15261 3271- AUUUUUACGGGUAACAGUGTT 1125CACUGUUACCCGUAAAAAUTT 1126 AD- 3289 15375 3274- UUUACGGGUAACAGUGAGGTT1127 CCUCACUGUUACCCGUAAATT 1128 AD- 3292 15262 3308-CAGACCAGGAAGCUCGGUGTT 1129 CACCGAGCUUCCUGGUCUGTT 1130 AD- 3326 153763310- GACCAGGAAGCUCGGUGAGTT 1131 CUCACCGAGCUUCCUGGUCTT 1132 AD- 332815377 3312- CCAGGAAGCUCGGUGAGUGTT 1133 CACUCACCGAGCUUCCUGGTT 1134 AD-3330 15409 3315- GGAAGCUCGGUGAGUGAUGTT 1135 CAUCACUCACCGAGCUUCCTT 1136AD- 3333 15378 3324- GUGAGUGAUGGCAGAACGATT 1137 UCGUUCUGCCAUCACUCACTT1138 AD- 3342 15410 3326- GAGUGAUGGCAGAACGAUGTT 1139CAUCGUUCUGCCAUCACUCTT 1140 AD- 3344 15379 3330- GAUGGCAGAACGAUGCCUGTT1141 CAGGCAUCGUUCUGCCAUCTT 1142 AD- 3348 15187 3336-AGAACGAUGCCUGCAGGCATT 1143 UGCCUGCAGGCAUCGUUCUTT 1144 AD- 3354 152633339- ACGAUGCCUGCAGGCAUGGTT 1145 CCAUGCCUGCAGGCAUCGUTT 1146 AD- 335715264 3348- GCAGGCAUGGAACUUUUUCTT 1147 GAAAAAGUUCCAUGCCUGCTT 1148 AD-3366 15297 3356- GGAACUUUUUCCGUUAUCATT 1149 UGAUAACGGAAAAAGUUCCTT 1150AD- 3374 15208 3357- GAACUUUUUCCGUUAUCACTT 1151 GUGAUAACGGAAAAAGUUCTT1152 AD- 3375 15209 3358- AACUUUUUCCGUUAUCACCTT 1153GGUGAUAACGGAAAAAGUUTT 1154 AD- 3376 15193 3370- UAUCACCCAGGCCUGAUUCTT1155 GAAUCAGGCCUGGGUGAUATT 1156 AD- 3388 15380 3378-AGGCCUGAUUCACUGGCCUTT 1157 AGGCCAGUGAAUCAGGCCUTT 1158 AD- 3396 152983383- UGAUUCACUGGCCUGGCGGTT 1159 CCGCCAGGCCAGUGAAUCATT 1160 AD- 340115299 3385- AUUCACUGGCCUGGCGGAGTT 1161 CUCCGCCAGGCCAGUGAAUTT 1162 AD-3403 15265 3406- GCUUCUAAGGCAUGGUCGGTT 1163 CCGACCAUGCCUUAGAAGCTT 1164AD- 3424 15381 3407- CUUCUAAGGCAUGGUCGGGTT 1165 CCCGACCAUGCCUUAGAAGTT1166 AD- 3425 15210 3429- GAGGGCCAACAACUGUCCCTT 1167GGGACAGUUGUUGGCCCUCTT 1168 AD- 3447 15270 3440- ACUGUCCCUCCUUGAGCACTsT1169 GUGCUCAAGGAGGGACAGUTsT 1170 AD- 3458 9591 3440-AcuGucccuccuuGAGcAcTsT 1171 GUGCUcAAGGAGGGAcAGUTsT 1172 AD- 3458 97173441- CUGUCCCUCCUUGAGCACCTsT 1173 GGUGCUCAAGGAGGGACAGTsT 1174 AD- 34599622 3441- cuGucccuccuuGAGcAccTsT 1175 GGUGCUcAAGGAGGGAcAGTsT 1176 AD-3459 9748 3480- ACAUUUAUCUUUUGGGUCUTsT 1177 AGACCCAAAAGAUAAAUGUTsT 1178AD- 3498 9587 3480- AcAuuuAucuuuuGGGucuTsT 1179 AGACCcAAAAGAuAAAUGUTsT1180 AD- 3498 9713 3480- AfcAfuUfuAfuCfuUfuUfgGfgUfcU 1181 p- 1182 AD-3498 fTsT aGfaCfcCfaAfaAfgAfuAfaAfuGfuTsT 14679 3480-ACfAUfUfUfAUfCfUfUfUfUfGGGUf 1183 AGACfCfCfAAAAGAUfAAAUfGUfTsT 1184 AD-3498 CfUfTsT 14689 3480- 1185 p- 1186 AD- 3498 AcAuUuAuCuUuUgGgUcUTsTaGfaCfcCfaAfaAfgAfuAfaAfuGfuTsT 14699 3480- AcAuUuAuCuUuUgGgUcUTsT 1187AGACfCfCfAAAAGAUfAAAUfGUfTsT 1188 AD- 3498 14709 3480-AfcAfuUfuAfuCfuUfuUfgGfgUfcU 1189 AGACCcaAAagAUAAAuguTsT 1190 AD- 3498fTsT 14719 3480- ACfAUfUfUfAUfCfUfUfUfUfGGGUf 1191AGACCcaAAagAUAAAuguTsT 1192 AD- 3498 CfUfTsT 14729 3480-AcAuUuAuCuUuUgGgUcUTsT 1193 AGACCcaAAagAUAAAuguTsT 1194 AD- 3498 147393480- GfcCfaUfcUfgCfuGfcCfgGfaGfcC 1195 p- 1196 AD- 3498 fTsTgGfcUfcCfgGfcAfgCfaGfaUfgGfcTsT 15085 3480- GCfCfAUfCfUfGCfUfGCfCfGGAGCf1197 GGCfUfCfCfGGCfAGCfAGAUfGGCfTsT 1198 AD- 3498 CfTsT 15095 3480-GcCaUcUgCuGcCgGaGcCTsT 1199 p- 1200 AD- 3498gGfcUfcCfgGfcAfgCfaGfaUfgGfcTsT 15105 3480- GcCaUcUgCuGcCgGaGcCTsT 1201GGCfUfCfCfGGCfAGCfAGAUfGGCfTsT 1202 AD- 3498 15115 3480-GfcCfaUfcUfgCfuGfcCfgGfaGfcC 1203 GGCUCauGCagCAGAUggcTsT 1204 AD- 3498fTsT 15125 3480- GCfCfAUfCfUfGCfUfGCfCfGGAGCf 1205GGCUCauGCagCAGAUggcTsT 1206 AD- 3498 CfTsT 15135 3480-GcCaUcUgCuGcCgGaGcCTsT 1207 GGCUCauGCagCAGAUggcTsT 1208 AD- 3498 151453481- CAUUUAUCUUUUGGGUCUGTsT 1209 CAGACCCAAAAGAUAAAUGTsT 1210 AD- 34999578 3481- cAuuuAucuuuuGGGucuGTsT 1211 cAGACCcAAAAGAuAAAUGTsT 1212 AD-3499 9704 3485- UAUCUUUUGGGUCUGUCCUTsT 1213 AGGACAGACCCAAAAGAUATsT 1214AD- 3503 9558 3485- uAucuuuuGGGucuGuccuTsT 1215 AGGAcAGACCcAAAAGAuATsT1216 AD- 3503 9684 3504- CUCUGUUGCCUUUUUACAGTsT 1217CUGUAAAAAGGCAACAGAGTsT 1218 AD- 3522 9634 3504- cucuGuuGccuuuuuAcAGTsT1219 CUGuAAAAAGGcAAcAGAGTsT 1220 AD- 3522 9760 3512-CCUUUUUACAGCCAACUUUTT 1221 AAAGUUGGCUGUAAAAAGGTT 1222 AD- 3530 154113521- AGCCAACUUUUCUAGACCUTT 1223 AGGUCUAGAAAAGUUGGCUTT 1224 AD- 353915266 3526- ACUUUUCUAGACCUGUUUUTT 1225 AAAACAGGUCUAGAAAAGUTT 1226 AD-3544 15382 3530- UUCUAGACCUGUUUUGCUUTsT 1227 AAGCAAAACAGGUCUAGAATsT 1228AD- 3548 9554 3530- uucuAGAccuGuuuuGcuuTsT 1229 AAGcAAAAcAGGUCuAGAATsT1230 AD- 3548 9680 3530- UfuCfuAfgAfcCfuGfuUfuUfgCfuU 1231 p- 1232 AD-3548 fTsT aAfgCfaAfaAfcAfgGfuCfuAfgAfaTsT 14676 3530-UfUfCfUfAGACfCfUfGUfUfUfUfGC 1233 AAGCfAAAACfAGGUfCfUfAGAATsT 1234 AD-3548 fUfUfTsT 14686 3530- UuCuAgAcCuGuUuUgCuUTsT 1235 p- 1236 AD- 3548aAfgCfaAfaAfcAfgGfuCfuAfgAfaTsT 14696 3530- UuCuAgAcCuGuUuUgCuUTsT 1237AAGCfAAAACfAGGUfCfUfAGAATsT 1238 AD- 3548 14706 3530-UfuCfuAfgAfcCfuGfuUfuUffCfuU 1239 AAGcAaaACagGUCUAgaaTsT 1240 AD- 3548fTsT 14716 3530- UfUfCfUfAGACfCfUfGUfUfUfUfGC 1241AAGcAaaACagGUCUAgaaTsT 1242 AD- 3548 fUfUfTsT 14726 3530-UuCuAgAcCuGuUuUgCuUTsT 1243 AAGcAaaACagGUCUAgaaTsT 1244 AD- 3548 147363530- CfaUfaGfgCfcUfgGfaGfuUfuAfuU 1245 p- 1246 AD- 3548 fTsTaAfuAfaAfcUfcCfaGfgCfcUfaUfgTsT 15082 3530- CfAUfAGGCfCfUfGGAGUfUfUfAUfU1247 AAUfAAACfUfCfCfAGGCfCfUfAUfGTsT 1248 AD- 3548 fTsT 15092 3530-CaUaGgCcUgGaGuUuAuUTsT 1249 p- 1250 AD- 3548aAfuAfaAfcUfcCfaGfgCfcUfaUfgTsT 15102 3530- CaUaGgCcUgGaGuUuAuUTsT 1251AAUfAAACfUfCfCfAGGCfCfUfAUfGTsT 1252 AD- 3548 15112 3530-CfaUfaGfgCfcUfgGfaGfuUfuAfuU 1253 AAUAAacUCcaGGCCUaugTsT 1254 AD- 3548fTsT 15122 3530- CfAUfAGGCfCfUfGGAGUfUfUfAUfU 1255AAUAAacUCcaGGCCUaugTsT 1256 AD- 3548 fTsT 15132 3530-CaUaGgCcUgGaGuUuAuUTsT 1257 AAUAAacUCcaGGCCUaugTsT 1258 AD- 3548 151423531- UCUAGACCUGUUUUGCUUUTsT 1259 AAAGCAAAACAGGUCUAGATsT 1260 AD- 35499553 3531- ucuAGAccuGuuuuGcuuuTsT 1261 AAAGcAAAAcAGGUCuAGATsT 1262 AD-3549 9679 3531- UfcUfaGfaCfcUfgUfuUfuGfcUfuU 1263 p- 1264 AD- 3549 fTsTaAfaGfcAfaAfaCfaGfgUfcUfaGfaTsT 14675 3531- UfCfUfAGACfCfUfGUfUfUfUfGCfU1265 AAAGCfAAAACfAGGUfCfUfAGATsT 1266 AD- 3549 fUfUfTsT 14685 3531-UcUaGaCcUgUuUuGcUuUTsT 1267 p- 1268 AD- 3549aAfaGfcAfaAfaCfaGfgUfcUfaGfaTsT 14695 3531- UcUaGaCcUgUuUuGcUuUTsT 1269AAAGCfAAAACfAGGUfCfUfAGATsT 1270 AD- 3549 14705 3531-UfcUfaGfaCfcUfgUfuUfuGfcUfuU 1271 AAAGCaaAAcaGGUCUagaTsT 1272 AD- 3549fTsT 14715 3531- UfCfUfAGACfCfUfGUfUfUfUfGCfU 1273AAAGCaaAAcaGGUCUagaTsT 1274 AD- 3549 fUfUfTsT 14725 3531-UcUaGaCcUgUuUuGcUuUTsT 1275 AAAGCaaAAcaGGUCUagaTsT 1276 AD- 3549 147353531- UfcAfuAfgGfcCfuGfgAfgUfuUfaU 1277 p- 1278 AD- 3549 fTsTaUfaAfaCfuCfcAfgGfcCfuAfuGfaTsT 15081 3531- UfCfAUfAGGCfCfUfGGAGUfUfUfAU1279 AUfAAACfUfCfCfAGGCfCfUfAUfGATsT 1280 AD- 3549 fTsT 15091 3531-UcAuAgGcCuGgAgUuUaUTsT 1281 p- 1282 AD- 3549aUfaAfaCfuCfcAfgGfcCfuAfuGfaTsT 15101 3531- UcAuAgGcCuGgAgUuUaUTsT 1283AUfAAACfUfCfCfAGGCfCfUfAUfGATsT 1284 AD- 3549 15111 3531-UfcAfuAfgGfcCfuGfgAfgUfuUfaU 1285 AUAAAcuCCagGCCUAugaTsT 1286 AD- 3549fTsT 15121 3531- UfCfAUfAGGCfCfUfGGAGUfUfUfAU 1287AUAAAcuCCagGCCUAugaTsT 1288 AD- 3549 fTsT 15131 3531-UcAuAgGcCuGgAgUuUaUTsT 1289 AUAAAcuCCagGCCUAugaTsT 1290 AD- 3549 151413557- UGAAGAUAUUUAUUCUGGGTsT 1291 CCCAGAAUAAAUAUCUUCATsT 1292 AD- 35759626 3557- uGAAGAuAuuuAuucuGGGTsT 1293 CCcAGAAuAAAuAUCUUcATsT 1294 AD-3575 9752 3570- UCUGGGUUUUGUAGCAUUUTsT 1295 AAAUGCUACAAAACCCAGATsT 1296AD- 3588 9629 3570- ucuGGGuuuuGuAGcAuuuTsT 1297 AAAUGCuAcAAAACCcAGATsT1298 AD- 3588 9755 3613- AUAAAAACAAACAAACGUUTT 1299AACGUUUGUUUGUUUUUAUTT 1300 AD- 3631 15412 3617- AAACAAACAAACGUUGUCCTT1301 GGACAACGUUUGUUUGUUUTT 1302 AD- 3635 15211 3618-AACAAACAAACGUUGUCCUTT 1303 AGGACAACGUUUGUUUGUUTT 1304 AD- 3636 15300 U,C, A, G: corresponding ribonucleotide; T: deoxythymidine; u, c, a, g:corresponding 2′-O-methyl ribonucleotide; Uf, Cf, Af, Gf: corresponding2′-deoxy-2′-fluoro ribonucleotide; where nucleotides are written insequence, they are connected by 3′-5′ phosphodiester groups; nucleotideswith interjected “s” are connected by 3′-O-5′-O phosphorothiodiestergroups; unless denoted by prefix “p-”, oligonucleotides are devoid of a5′-phosphate group on the 5′-most nucleotide; all oligonucleotides bear3′-OH on the 3′-most nucleotide

TABLE 1b Screening of siRNAs targeted to PCSK9 Mean percent remainingmRNA transcript at IC50 in siRNA concentration/in cell type Cynomolgous100 30 IC50 in monkey Duplex nM/ 30 nM/ 3 nM/ nM/ HepG2 Hepatocyte nameHepG2 HepG2 HepG2 HeLa [nM] [nM]s AD-15220 35 AD-15275 56 AD-15301 70AD-15276 42 AD-15302 32 AD-15303 37 AD-15221 30 AD-15413 61 AD-15304 70AD-15305 36 AD-15306 20 AD-15307 38 AD-15277 50 AD-9526 74 89 AD-9652 97AD-9519 78 AD-9645 66 AD-9523 55 AD-9649 60 AD-9569 112 AD-9695 102AD-15222 75 AD-15278 78 AD-15178 83 AD-15308 84 AD-15223 67 AD-15309 34AD-15279 44 AD-15194 63 AD-15310 42 AD-15311 30 AD-15392 18 AD-15312 21AD-15313 19 AD-15280 81 AD-15267 82 AD-15314 32 AD-15315 74 AD-9624 94AD-9750 96 AD-9623 43 66 AD-9749 105 AD-15384 48 AD-9607 32 28 0.20AD-9733 78 73 AD-9524 23 28 0.07 AD-9650 91 90 AD-9520 23 32 AD-9520 23AD-9646 97 108 AD-9608 37 AD-9734 91 AD-9546 32 AD-9672 57 AD-15385 54AD-15393 31 AD-15316 37 AD-15317 37 AD-15318 63 AD-15195 45 AD-15224 57AD-15188 42 AD-15225 51 AD-15281 89 AD-15282 75 AD-15319 61 AD-15226 56AD-15271 25 AD-15283 25 AD-15284 64 AD-15189 17 AD-15227 62 AD-9547 3129 0.20 AD-9673 56 57 AD-9548 54 60 AD-9674 36 57 AD-9529 60 AD-9655 140AD-9605 27 31 0.27 AD-9731 31 31 0.32 AD-9596 37 AD-9722 76 AD-9583 42AD-9709 104 AD-9579 113 AD-9705 81 AD-15394 32 AD-15196 72 AD-15197 85AD-15198 71 AD-9609 66 71 AD-9735 115 AD-9537 145 AD-9663 102 AD-9528113 AD-9654 107 AD-9515 49 AD-9641 92 AD-9514 57 AD-9640 89 AD-9530 75AD-9656 77 AD-9538 79 80 AD-9664 53 AD-9598 69 83 AD-9724 127 AD-9625 5888 AD-9751 60 AD-9556 46 AD-9682 38 AD-9539 56 63 AD-9665 83 AD-9517 36AD-9643 40 AD-9610 36 34 0.04 AD-9736 22 29 0.04 0.5 AD-14681 33AD-14691 27 AD-14701 32 AD-14711 33 AD-14721 22 AD-14731 21 AD-14741 22AD-15087 37 AD-15097 51 AD-15107 26 AD-15117 28 AD-15127 33 AD-15137 54AD-15147 52 AD-9516 94 AD-9642 105 AD-9562 46 51 AD-9688 26 34 4.20AD-14677 38 AD-14687 52 AD-14697 35 AD-14707 58 AD-14717 42 AD-14727 50AD-14737 32 AD-15083 16 AD-15093 24 AD-15103 11 AD-15113 34 AD-15123 19AD-15133 15 AD-15143 16 AD-9521 50 AD-9647 62 AD-9611 48 AD-9737 68AD-9592 46 55 AD-9718 78 AD-9561 64 AD-9687 84 AD-9636 42 41 2.10AD-9762 9 28 0.40 0.5 AD-9540 45 AD-9666 81 AD-9535 48 73 AD-9661 83AD-9559 35 AD-9685 77 AD-9533 100 AD-9659 88 AD-9612 122 AD-9738 83AD-9557 75 96 AD-9683 48 AD-9531 31 32 0.53 AD-9657 23 29 0.66 0.5AD-14673 81 AD-14683 56 AD-14693 56 AD-14703 68 AD-14713 55 AD-14723 24AD-14733 34 AD-15079 85 AD-15089 54 AD-15099 70 AD-15109 67 AD-15119 67AD-15129 57 AD-15139 69 AD-9542 160 AD-9668 92 AD-9739 109 AD-9637 56 83AD-9763 79 AD-9630 82 AD-9756 63 AD-9593 55 AD-9719 115 AD-9601 111AD-9727 118 AD-9573 36 42 1.60 AD-9699 32 36 2.50 AD-15228 26 AD-1539553 AD-9602 126 AD-9728 94 AD-15386 45 AD-9580 112 AD-9706 86 AD-9581 35AD-9707 81 AD-9543 51 AD-9669 97 AD-9574 74 AD-9700 AD-15320 26 AD-1532134 AD-15199 64 AD-15167 86 AD-15164 41 AD-15166 43 AD-15322 64 AD-1520046 AD-15213 27 AD-15229 44 AD-15215 49 AD-15214 101 AD-9315 15 32 0.98AD-9326 35 51 AD-9318 14 37 0.40 AD-9323 14 33 AD-9314 11 22 0.04AD-10792 0.10 0.10 AD-10796 0.1 0.1 AD-9638 101 AD-9764 112 AD-9525 53AD-9651 58 AD-9560 97 AD-9686 111 AD-9536 157 AD-9662 81 AD-9584 52 68AD-9710 111 AD-15323 62 AD-9551 91 AD-9677 62 AD-15230 52 AD-15231 25AD-15285 36 AD-15396 27 AD-15397 56 AD-9600 112 AD-9726 95 AD-9606 107AD-9732 105 AD-9633 56 75 AD-9759 111 AD-9588 66 AD-9714 106 AD-9589 6785 AD-9715 113 AD-9575 120 AD-9701 100 AD-9563 103 AD-9689 81 AD-9594 8095 AD-9720 92 AD-9585 83 AD-9711 122 AD-9614 100 AD-9740 198 AD-9615 116AD-9741 130 AD-9534 32 30 AD-9534 32 AD-9660 89 79 AD-15324 46 AD-1523219 AD-15233 25 AD-15234 59 AD-15286 109 AD-9590 122 AD-9716 114 AD-963234 AD-9758 96 AD-9567 41 AD-9693 50 AD-9586 81 104 AD-9712 107 AD-9564120 AD-9690 92 AD-9616 74 84 AD-9742 127 AD-15398 24 AD-9617 111 AD-9743104 AD-9635 73 90 AD-9761 15 33 0.5 AD-9568 76 AD-9694 52 AD-9576 47AD-9702 79 AD-9627 69 AD-9753 127 AD-9628 141 AD-9754 89 AD-9631 80AD-9757 78 AD-9595 31 32 AD-9721 87 70 AD-9544 68 AD-9670 67 AD-15235 25AD-15236 73 AD-15168 100 AD-15174 92 AD-15325 81 AD-15326 65 AD-9570 3542 AD-9696 77 AD-9566 38 AD-9692 78 AD-9532 100 AD-9658 102 AD-9549 50AD-9675 78 AD-9541 43 AD-9667 73 AD-9550 36 AD-9676 100 AD-9571 27 32AD-9697 74 89 AD-9572 47 53 AD-9698 73 AD-15327 82 AD-9639 30 35 AD-976582 74 AD-9518 31 35 0.60 AD-9518 31 AD-9644 35 37 2.60 0.5 AD-14672 26AD-14682 27 AD-14692 22 AD-14702 19 AD-14712 25 AD-14722 18 AD-14732 32AD-15078 86 AD-15088 97 AD-15098 74 AD-15108 67 AD-15118 76 AD-15128 86AD-15138 74 AD-15237 30 AD-15287 30 AD-15238 36 AD-15328 35 AD-15399 47AD-9582 37 AD-9708 81 AD-9545 31 43 AD-9671 15 33 2.50 AD-14674 16AD-14684 26 AD-14694 18 AD-14704 27 AD-14714 20 AD-14724 18 AD-14734 18AD-15080 29 AD-15090 23 AD-15100 26 AD-15110 23 AD-15120 20 AD-15130 20AD-15140 19 AD-9522 59 AD-9648 78 AD-9552 80 AD-9678 76 AD-9618 90AD-9744 91 AD-15239 38 AD-15212 19 AD-15240 43 AD-15177 59 AD-15179 13AD-15180 15 AD-15241 14 AD-15268 42 AD-15242 21 AD-15216 28 AD-15176 35AD-15181 35 AD-15243 22 AD-15182 42 AD-15244 31 AD-15387 23 AD-15245 18AD-9555 34 AD-9681 55 AD-9619 42 61 AD-9745 56 AD-9620 44 77 AD-9746 89AD-15288 19 AD-15246 16 AD-15289 37 AD-9324 59 67 AD-15329 103 AD-1533062 AD-15169 22 AD-15201 6 AD-15331 14 AD-15190 47 AD-15247 61 AD-1524822 AD-15175 45 AD-15249 51 AD-15250 96 AD-15400 12 AD-15332 22 AD-1538830 AD-15333 20 AD-15334 96 AD-15335 75 AD-15183 16 AD-15202 41 AD-1520339 AD-15272 49 AD-15217 16 AD-15290 15 AD-15218 13 AD-15389 13 AD-1533640 AD-15337 19 AD-15191 33 AD-15390 25 AD-15338 9 AD-15204 33 AD-1525176 AD-15205 14 AD-15171 16 AD-15252 58 AD-15339 20 AD-15253 15 AD-1534018 AD-15291 17 AD-15341 11 AD-15401 13 AD-15342 30 AD-15343 21 AD-1529216 AD-15344 20 AD-15254 18 AD-15345 18 AD-15206 15 AD-15346 16 AD-1534762 AD-9577 33 31 AD-9703 17 26 1 AD-14678 22 AD-14688 23 AD-14698 23AD-14708 14 AD-14718 31 AD-14728 25 AD-14738 31 AD-15084 19 AD-15094 11AD-15104 16 AD-15114 15 AD-15124 11 AD-15134 12 AD-15144 9 AD-15391 7AD-15348 13 AD-15349 8 AD-15170 40 AD-15350 14 AD-15402 27 AD-15293 27AD-15351 14 AD-15403 11 AD-15404 38 AD-15207 15 AD-15352 23 AD-15255 31AD-9603 123 AD-9729 56 AD-9599 139 AD-9725 38 AD-9621 77 AD-9747 63AD-15405 32 AD-15353 39 AD-15354 49 AD-15406 35 AD-15407 39 AD-15355 18AD-15356 50 AD-15357 54 AD-15269 23 AD-9565 74 AD-9691 49 AD-15358 12AD-15359 24 AD-15360 13 AD-15219 19 AD-15361 24 AD-15273 36 AD-15362 31AD-15192 20 AD-15256 19 AD-15363 33 AD-15364 24 AD-9604 35 49 AD-9730 85AD-9527 45 AD-9653 86 AD-15365 62 AD-15294 30 AD-15173 12 AD-15366 21AD-15367 11 AD-15257 18 AD-15184 50 AD-15185 12 AD-15258 73 AD-15186 36AD-15274 19 AD-15368 7 AD-15369 17 AD-15370 19 AD-15259 38 AD-15408 52AD-9597 23 21 0.04 AD-9723 12 26 0.5 AD-14680 15 AD-14690 18 AD-14700 15AD-14710 15 AD-14720 18 AD-14730 18 AD-14740 17 AD-15086 85 AD-15096 70AD-15106 71 AD-15116 73 AD-15126 71 AD-15136 56 AD-15146 72 AD-15260 79AD-15371 24 AD-15372 52 AD-15172 27 AD-15295 22 AD-15373 11 AD-15163 18AD-15165 13 AD-15374 23 AD-15296 13 AD-15261 20 AD-15375 90 AD-15262 72AD-15376 14 AD-15377 19 AD-15409 17 AD-15378 18 AD-15410 8 AD-15379 11AD-15187 36 AD-15263 18 AD-15264 75 AD-15297 21 AD-15208 6 AD-15209 28AD-15193 131 AD-15380 88 AD-15298 43 AD-15299 99 AD-15265 95 AD-15381 18AD-15210 40 AD-15270 83 AD-9591 75 95 AD-9717 105 AD-9622 94 AD-9748 103AD-9587 63 49 AD-9713 22 25 0.5 AD-14679 19 AD-14689 24 AD-14699 19AD-14709 21 AD-14719 24 AD-14729 23 AD-14739 24 AD-15085 74 AD-15095 60AD-15105 33 AD-15115 30 AD-15125 54 AD-15135 51 AD-15145 49 AD-9578 4961 AD-9704 111 AD-9558 66 AD-9684 63 AD-9634 29 30 AD-9760 14 27AD-15411 5 AD-15266 23 AD-15382 12 AD-9554 23 24 AD-9680 12 22 0.1 0.1AD-14676 12 .1 AD-14686 13 AD-14696 12 .1 AD-14706 18 .1 AD-14716 17 .1AD-14726 16 .1 AD-14736 9 .1 AD-15082 27 AD-15092 28 AD-15102 19AD-15112 17 AD-15122 56 AD-15132 39 AD-15142 46 AD-9553 27 22 0.02AD-9679 17 21 0.1 AD-14675 11 AD-14685 19 AD-14695 12 AD-14705 16AD-14715 19 AD-14725 19 AD-14735 19 AD-15081 30 AD-15091 16 AD-15101 16AD-15111 11 AD-15121 19 AD-15131 17 AD-15141 18 AD-9626 97 68 AD-9752 2833 AD-9629 23 24 AD-9755 28 29 0.5 AD-15412 21 AD-15211 73 AD-15300 41

TABLE 2a Sequences of modified dsRNA targeted to PCSK9 SEQ SEQ Duplex IDID number Sense strand sequence (5′-3′)¹ NO:Antisense-strand sequence (5′-3′)¹ NO: AD-10792 GccuGGAGuuuAuucGGAATsT1305 UUCCGAAuAAACUCcAGGCTsT 1306 AD-10793 GccuGGAGuuuAuucGGAATsT 1307uUcCGAAuAAACUccAGGCTsT 1308 AD-10796 GccuGGAGuuuAuucGGAATsT 1309UUCCGAAUAAACUCCAGGCTsT 1310 AD-12038 GccuGGAGuuuAuucGGAATsT 1311uUCCGAAUAAACUCCAGGCTsT 1312 AD-12039 GccuGGAGuuuAuucGGAATsT 1313UuCCGAAUAAACUCCAGGCTsT 1314 AD-12040 GccuGGAGuuuAuucGGAATsT 1315UUcCGAAUAAACUCCAGGCTsT 1316 AD-12041 GccuGGAGuuuAuucGGAATsT 1317UUCCGAAUAAACUCCAGGCTsT 1318 AD-12042 GCCUGGAGUUUAUUCGGAATsT 1319uUCCGAAUAAACUCCAGGCTsT 1320 AD-12043 GCCUGGAGUUUAUUCGGAATsT 1321UuCCGAAUAAACUCCAGGCTsT 1322 AD-12044 GCCUGGAGUUUAUUCGGAATsT 1323UUcCGAAUAAACUCCAGGCTsT 1324 AD-12045 GCCUGGAGUUUAUUCGGAATsT 1325UUCCGAAUAAACUCCAGGCTsT 1326 AD-12046 GccuGGAGuuuAuucGGAA 1327UUCCGAAUAAACUCCAGGCscsu 1328 AD-12047 GccuGGAGuuuAuucGGAAA 1329UUUCCGAAUAAACUCCAGGCscsu 1330 AD-12048 GccuGGAGuuuAuucGGAAAA 1331UUUUCCGAAUAAACUCCAGGCscsu 1332 AD-12049 GccuGGAGuuuAuucGGAAAAG 1333CUUUUCCGAAUAAACUCCAGGCscsu 1334 AD-12050 GccuGGAGuuuAuucGGAATTab 1335UUCCGAAUAAACUCCAGGCTTab 1336 AD-12051 GccuGGAGuuuAuucGGAAATTab 1337UUUCCGAAuAAACUCCAGGCTTab 1338 AD-12052 GccuGGAGuuuAuucGGAAAATTab 1339UUUUCCGAAUAAACUCCAGGCTTab 1340 AD-12053 GccuGGAGuuuAuucGGAAAAGTTab 1341CUUUUCCGAAUAAACUCCAGGCTTab 1342 AD-12054 GCCUGGAGUUUAUUCGGAATsT 1343UUCCGAAUAAACUCCAGGCscsu 1344 AD-12055 GccuGGAGuuuAuucGGAATsT 1345UUCCGAAUAAACUCCAGGCscsu 1346 AD-12056 GcCuGgAgUuUaUuCgGaA 1347UUCCGAAUAAACUCCAGGCTTab 1348 AD-12057 GcCuGgAgUuUaUuCgGaA 1349UUCCGAAUAAACUCCAGGCTsT 1350 AD-12058 GcCuGgAgUuUaUuCgGaA 1351UUCCGAAuAAACUCcAGGCTsT 1352 AD-12059 GcCuGgAgUuUaUuCgGaA 1353uUcCGAAuAAACUccAGGCTsT 1354 AD-12060 GcCuGgAgUuUaUuCgGaA 1355UUCCGaaUAaaCUCCAggc 1356 AD-12061 GcCuGgnAgUuUaUuCgGaATsT 1357UUCCGaaUAaaCUCCAggcTsT 1358 AD-12062 GcCuGgAgUuUaUuCgGaATTab 1359UUCCGaaUAaaCUCCAggcTTab 1360 AD-12063 GcCuGgAgUuUaUuCgGaA 1361UUCCGaaUAaaCUCCAggcscsu 1362 AD-12064 GcCuGgnAgUuUaUuCgGaATsT 1363UUCCGAAuAAACUCcAGGCTsT 1364 AD-12065 GcCuGgAgUuUaUuCgGaATTab 1365UUCCGAAuAAACUCcAGGCTTab 1366 AD-12066 GcCuGgAgUuUaUuCgGaA 1367UUCCGAAuAAACUCcAGGCscsu 1368 AD-12067 GcCuGgnAgUuUaUuCgGaATsT 1369UUCCGAAUAAACUCCAGGCTsT 1370 AD-12068 GcCuGgAgUuUaUuCgGaATTab 1371UUCCGAAUAAACUCCAGGCTTab 1372 AD-12069 GcCuGgAgUuUaUuCgGaA 1373UUCCGAAUAAACUCCAGGCscsu 1374 AD-12338 GfcCfuGfgAfgUfuUfaUfuCfgGfaAf 1375P-uUfcCfgAfaUfaAfaCfuCfcAfgGfc 1376 AD-12339 GcCuGgAgUuUaUuCgGaA 1377P-uUfcCfgAfaUfaAfaCfuCfcAfgGfc 1378 AD-12340 GccuGGAGuuuAuucGGAA 1379P-uUfcCfgAfaUfaAfaCfuCfcAfgGfc 1380 AD-12341GfcCfuGfgAfgUfuUfaUfuCfgGfaAffsT 1381 P-uUfcCfgAfaUfaAfaCfuCfcAfgGfcTsT1382 AD-12342 GfcCfuGfgAfgUfuUfaUfuCfgGfaAffsT 1383UUCCGAAuAAACUCcAGGCTsT 1384 AD-12343 GfcCfuGfgAfgUfuUfaUfuCfgGfaAffsT1385 uUcCGAAuAAACUccAGGCTsT 1386 AD-12344GfcCfuGfgAfgUfuUfaUfuCfgGfaAffsT 1387 UUCCGAAUAAACUCCAGGCTsT 1388AD-12345 GfcCfuGfgAfgUfuUfaUfuCfgGfaAffsT 1389 UUCCGAAUAAACUCCAGGCscsu1390 AD-12346 GfcCfuGfgAfgUfuUfaUfuCfgGfaAffsT 1391UUCCGaaUAaaCUCCAggcscsu 1392 AD-12347 GCCUGGAGUUUAUUCGGAATsT 1393P-uUfcCfgAfaUfaAfaCfuCfcAfgGfcTsT 1394 AD-12348 GccuGGAGuuuAuucGGAATsT1395 P-uUfcCfgAfaUfaAfaCfuCfcAfgGfcTsT 1396 AD-12349GcCuGgnAgUuUaUuCgGaATsT 1397 P-uUfcCfgAfaUfaAfaCfuCfcAfgGfcTsT 1398AD-12350 GfcCfuGfgAfgUfuUfaUfuCfgGfaAfTTab 1399P-uUfcCfgAfaUfaAfaCfuCfcAfgGfcTTab 1400 AD-12351GfcCfuGfgAfgUfuUfaUfuCfgGfaAf 1401 P-uUfcCfgAfaUfaAfaCfuCfcAfgGfcsCfsu1402 AD-12352 GfcCfuGfgAfgUfuUfaUfuCfgGfaAf 1403 UUCCGaaUAaaCUCCAggcscsu1404 AD-12354 GfcCfuGfgAfgUfuUfaUfuCfgGfaAf 1405 UUCCGAAUAAACUCCAGGCscsu1406 AD-12355 GfcCfuGfgAfgUfuUfaUfuCfgGfaAf 1407 UUCCGAAuAAACUCcAGGCTsT1408 AD-12356 GfcCfuGfgAfgUfuUfaUfuCfgGfaAf 1409 uUcCGAAuAAACUccAGGCTsT1410 AD-12357 GmocCmouGmogAm02gUmouUmoaUmouCm 1411 UUCCGaaUAaaCUCCAggc1412 ogGmoaA AD-12358 GmocCmouGmogAm02gUmouUmoaUmouCm 1413P-uUfcCfgAfaUfaAfaCfuCfcAfgGfc 1414 ogGmoaA AD-12359GmocCmouGmogAm02gUmouUmoaUmouCm 1415 P-uUfcCfgAfaUfaAfaCfuCfcAfgGfcsCfsu1416 ogGmoaA AD-12360 GmocCmouGmogAm02gUmouUmoaUmouCm 1417UUCCGAAUAAACUCCAGGCscsu 1418 ogGmoaA AD-12361GmocCmouGmogAm02gUmouUmoaUmouCm 1419 UUCCGAAuAAACUCcAGGCTsT 1420 ogGmoaAAD-12362 GmocCmouGmogAm02gUmouUmoaUmouCm 1421 uUcCGAAuAAACUccAGGCTsT1422 ogGmoaA AD-12363 GmocCmouGmogAm02gUmouUmoaUmouCm 1423UUCCGaaUAaaCUCCAggcscsu 1424 ogGmoaA AD-12364GmocCmouGmogAmogUmouUmoaUmouCmo 1425 UUCCGaaUAaaCUCCAggcTsT 1426gGmoaATsT AD-12365 GmocCmouGmogAmogUmouUmoaUmouCmo 1427UUCCGAAuAAACUCcAGGCTsT 1428 gGmoaATsT AD-12366GmocCmouGmogAmogUmouUmoaUmouCmo 1429 UUCCGAAUAAACUCCAGGCTsT 1430gGmoaATsT AD-12367 GmocmocmouGGAGmoumoumouAmoumoum 1431UUCCGaaUAaaCUCCAggcTsT 1432 ocGGAATsT AD-12368GmocmocmouGGAGmoumoumouAmoumoum 1433 UUCCGAAuAAACUCcAGGCTsT 1434ocGGAATsT AD-12369 GmocmocmouGGAGmoumoumouAmoumoum 1435UUCCGAAUAAACUCCAGGCTsT 1436 ocGGAATsT AD-12370GmocmocmouGGAGmoumoumouAmoumoum 1437 P-UfUfCfCfGAAUfAAACfUfCfCfAGGCfTsT1438 ocGGAATsT AD-12371 GmocmocmouGGAGmoumoumouAmoumoum 1439P-UfUfCfCfGAAUfAAACfUfCfCfAGGCfsCfsUf 1440 ocGGAATsT AD-12372GmocmocmouGGAGmoumoumouAmoumoum 1441 P-uUfcCfgAfaUfaAfaCfuCfcAfgGfcsCfsu1442 ocGGAATsT AD-12373 GmocmocmouGGAGmoumoumouAmoumoum 1443UUCCGAAUAAACUCCAGGCTsT 1444 ocGGAATsT AD-12374GCfCfUfGGAGUfUfUfAUfUfCfGGAATsT 1445 UfUfCfCfGAAUfAAACfUfCfCfAGGCfTsT1446 AD-12375 GCfCfUfGGAGUfUfUfAUfUfCfGGAATsT 1447UUCCGAAUAAACUCCAGGCTsT 1448 AD-12377 GCfCfUfGGAGUfUfUfAUfUfCfGGAATsT1449 uUcCGAAuAAACUccAGGCTsT 1450 AD-12378GCfCfUfGGAGUfUfUfAUfUfCfGGAATsT 1451 UUCCGaaUAaaCUCCAggcscsu 1452AD-12379 GCfCfUfGGAGUfUfUfAUfUfCfGGAATsT 1453 UUCCGAAUAAACUCCAGGCscsu1454 AD-12380 GCfCfUfGGAGUfUfUfAUfUfCfGGAATsT 1455P-uUfcCfgAfaUfaAfaCfuCfcAfgGfcsCfsu 1456 AD-12381GCfCfUfGGAGUfUfUfAUfUfCfGGAATsT 1457 P-uUfcCfgAfaUfaAfaCfuCfcAfgGfcTsT1458 AD-12382 GCfCfUfGGAGUfUfUfAUfUfCfGGAATsT 1459P-UfUfCfCfGAAUfAAACfUfCfCfAGGCfTsT 1460 AD-12383 GCCUGGAGUUUAUUCGGAATsT1461 P-UfUfCfCfGAAUfAAACfUfCfCfAGGCfTsT 1462 AD-12384GccuGGAGuuuAuucGGAATsT 1463 P-UfUfCfCfGAAUfAAACfUfCfCfAGGCfTsT 1464AD-12385 GcCuGgnAgUuUaUuCgGaATsT 1465 P-UfUfCfCfGAAUfAAACfUfCfCfAGGCfTsT1466 AD-12386 GfcCfuGfgAfgUfuUfaUfuCfgGfaAf 1467P-UfUfCfCfGAAUfAAACfUfCfCfAGGCfTsT 1468 AD-12387GCfCfUfGGAGGUfUfUfAUfUfCfGGAA 1469 UfUfCfCfGAAUfAAACfUfCfCfAGGCfsCfsUf1470 AD-12388 GCfCfUfGGAGGUfUfUfAUfUfCfGGAA 1471P-uUfcCfgAfaUfaAfaCfuCfcAfgGfc 1472 AD-12389GCfCfUfGGAGGUfUfUfAUfUfCfGGAA 1473 P-uUfcCfgAfaUfaAfaCfuCfcAfgGfcsCfsu1474 AD-12390 GCfCfUfGGAGGUfUfUfAUfUfCfGGAA 1475 UUCCGAAUAAACUCCAGGCscsu1476 AD-12391 GCfCfUfGGAGGUfUfUfAUfUfCfGGAA 1477 UUCCGaaUAaaCUCCAggc1478 AD-12392 GCfCfUfGGAGGUfUfUfAUfUfCfGGAA 1479 UUCCGAAUAAACUCCAGGCTsT1480 AD-12393 GCfCfUfGGAGGUfUfUfAUfUfCfGGAA 1481 UUCCGAAuAAACUCcAGGCTsT1482 AD-12394 GCfCfUfGGAGGUfUfUfAUfUfCfGGAA 1483 uUcCGAAuAAACUccAGGCTsT1484 AD-12395 GmocCmouGmogAmogUmouUmoaUmouCmo 1485P-UfUfCfCfGAAUfAAACfUfCfCfAGGCfsCfsUf 1486 gGmoaATsT AD-12396GmocCmouGmogAm02gUmouUmoaUmouCm 1487P-UfUfCfCfGAAUfAAACfUfCfCfAGGCfsCfsUf 1488 ogGmoaA AD-12397GfcCfuGfgAfgUfuUfaUfuCfgGfaAf 1489 P-UfUfCfCfGAAUfAAACfUfCfCfAGGCfsCfsUf1490 AD-12398 GfcCfuGfgAfgUfuUfaUfuCfgGfaAffsT 1491P-UfUfCfCfGAAUfAAACfUfCfCfAGGCfsCfsUf 1492 AD-12399GcCuGgnAgUuUaUuCgGaATsT 1493 P-UfUfCfCfGAAUfAAACfUfCfCfAGGCfsCfsUf 1494AD-12400 GCCUGGAGUUUAUUCGGAATsT 1495P-UfUfCfCfGAAUfAAACfUfCfCfAGGCfsCfsUf 1496 AD-12401GccuGGAGuuuAuucGGAATsT 1497 P-UfUfCfCfGAAUfAAACfUfCfCfAGGCfsCfsUf 1498AD-12402 GccuGGAGuuuAuucGGAA 1499 P-UfUfCfCfGAAUfAAACfUfCfCfAGGCfsCfsUf1500 AD-12403 GCfCfUfGGAGGUfUfUfAUfUfCfGGAA 1501P-UfUfCfCfGAAUfAAACfUfCfCfAGGCfsCfsUf 1502 AD-9314GCCUGGAGUUUAUUCGGAATsT 1503 UUCCGAAUAAACUCCAGGCTsT 1504 AD-10794ucAuAGGccuGGAGuuuAudTsdT 1525 AuAAACUCcAGGCCuAUGAdTsdT 1526 AD-10795ucAuAGGccuGGAGuuuAudTsdT 1527 AuAAACUccAGGcCuAuGAdTsdT 1528 AD-10797ucAuAGGccuGGAGuuuAudTsdT 1529 AUAAACUCCAGGCCUAUGAdTsdT 1530 U, C, A, G:corresponding ribonucleotide; T: deoxythymidine; u, c, a, g:corresponding 2′-O-methyl ribonucleotide; Uf, Cf, Af, Gf: corresponding2′-deoxy-2′-fluoro ribonucleotide; moc, mou, mog, moa: corresponding2′-MOE nucleotide; where nucleotides are written in sequence, they areconnected by 3′-5′ phosphodiester groups; ab: 3′-terminal abasicnucleotide; nucleotides with interjected “s” are connected by 3′-O-5′-Ophosphorothiodiester groups; unless denoted by prefix “p-”,oligonucleotides are devoid of a 5′-phosphate group on the 5′-mostnucleotide; all oligonucleotides bear 3′-OH on the 3′-most nucleotide

TABLE 2b Screening of dsRNAs targeted to PCSK9 Remaining mRNA in % ofcontrols at Duplex number siRNA conc. of 30 nM AD-10792 15 AD-10793 32AD-10796 13 AD-12038 13 AD-12039 29 AD-12040 10 AD-12041 11 AD-12042 12AD-12043 13 AD-12044 7 AD-12045 8 AD-12046 13 AD-12047 17 AD-12048 43AD-12049 34 AD-12050 16 AD-12051 31 AD-12052 81 AD-12053 46 AD-12054 8AD-12055 13 AD-12056 11 AD-12057 8 AD-12058 9 AD-12059 23 AD-12060 10AD-12061 7 AD-12062 10 AD-12063 19 AD-12064 15 AD-12065 16 AD-12066 20AD-12067 17 AD-12068 18 AD-12069 13 AD-12338 15 AD-12339 14 AD-12340 19AD-12341 12 AD-12342 13 AD-12343 24 AD-12344 9 AD-12345 12 AD-12346 13AD-12347 11 AD-12348 8 AD-12349 11 AD-12350 17 AD-12351 11 AD-12352 11AD-12354 11 AD-12355 9 AD-12356 25 AD-12357 56 AD-12358 29 AD-12359 30AD-12360 15 AD-12361 20 AD-12362 51 AD-12363 11 AD-12364 25 AD-12365 18AD-12366 23 AD-12367 42 AD-12368 40 AD-12369 26 AD-12370 68 AD-12371 60AD-12372 60 AD-12373 55 AD-12374 9 AD-12375 16 AD-12377 88 AD-12378 6AD-12379 6 AD-12380 8 AD-12381 10 AD-12382 7 AD-12383 7 AD-12384 8AD-12385 8 AD-12386 11 AD-12387 13 AD-12388 19 AD-12389 16 AD-12390 17AD-12391 21 AD-12392 28 AD-12393 17 AD-12394 75 AD-12395 55 AD-12396 59AD-12397 20 AD-12398 11 AD-12399 13 AD-12400 12 AD-12401 13 AD-12402 14AD-12403 4 AD-9314 9

TABLE 3 Cholesterol levels of rats treated with LNP01-10792 Dosage of 5mg/kg, n = 6 rats per group Day Total serum cholesterol (relative to PBScontrol) 2 0.329 ± 0.035 4 0.350 ± 0.055 7 0.402 ± 0.09  9 0.381 ± 0.06111 0.487 ± 0.028 14 0.587 ± 0.049 16 0.635 ± 0.107 18 0.704 ± 0.060 210.775 ± 0.102 28 0.815 ± 0.103

TABLE 4 Serum LDL-C levels of cynomolgus monkeys treated with LNPformulated dsRNAs Serum LDL-C (relative to pre-dose) Day 3 Day 4 Day 5Day 7 Day 14 Day 21 PBS 1.053 ± 0.158 0.965 ± 0.074 1.033 ± 0.085 1.033± 0.157 1.009 ± 0.034 n = 3 LNP01-1955 1.027 ± 0.068 1.104 ± 0.114 n = 3LNP01-10792 0.503 ± 0.055 0.596 ± 0.111 0.674 ± 0.139 0.644 ± 0.1210.958 ± 0.165 1.111 ± 0.172 n = 5 LNP01-9680 0.542 ± 0.155 0.437 ± 0.0760.505 ± 0.071 0.469 ± 0.066 0.596 ± 0.080 0.787 ± 0.138 n = 4

TABLE 5a Modified dsRNA targeted to PCSK9 Position SEQ in human ID Nameaccess.# Sense Antisense Sequence 5′-3′ NO: AD- 1091 unmodifiedunmodified GCCUGGAGUUUAUUCGGAAdTdT 1505 1a1 UUCCGAAUAAACUCCAGGCdTsdT1506 AD- 1091 2′OMe 2′OMe GccuGGAGuuuAuucGGAAdTsdT 1507 1a2UUCCGAAuAAACUCcAGGCdTsdT 1508 AD- 1091 Alt 2′F, Alt 2′F,GfcCfuGfgAfgUfuUfaUfuCfgGfaAfdTdT 1509 1a3 2′OMe 2′OMepuUfcCfgAfaUfaAfaCfuCfcAfgGfcdTsdT 1510 AD- 1091 2′OMe 2′F all Py,GccuGGAGuuuAuucGGAAdTsdT 1511 1a4 5′PhosphatePUfUfCfCfGAAUfAAACfUfCfCfAGGCfdTsdT 1512 AD- 1091 2′F 2′F all Py,GCfCfUfGGAGUfUfUfAUfUfCfGGAAdTsdT 1513 1a5 5′PhosphatePUfUfCfCfGAAUfAAACfUfCfCfAGGCfdTsdT 1514 AD-2a1 3530 2′OMe 2′OMeuucuAGAccuGuuuuGcuudTsdT 1515 (3′UTR) AAGcAAAAcAGGUCuAGAAdTsdT 1516AD-3a1  833 2′OMe 2′OMe AGGuGuAucuccuAGAcAcdTsdT 1517GUGUCuAGGAGAuAcACCUdTsdT 1518 AD- N/A 2′OMe 2′OMecuuAcGcuGAGuAcuucGAdTsdT 1519 ctrl UCGAAGuACUcAGCGuAAGdTsdT 1520 (Luc.)U, C, A, G: corresponding ribonucleotide; T: deoxythymidine; u, c, a, g:corresponding 2′-O-methyl ribonucleotide; Uf, Cf, Af, Gf: corresponding2′-deoxy-2′-fluoro ribonucleotide; where nucleotides are written insequence, they are connected by 3′-5′ phosphodiester groups; nucleotideswith interjected “s” are connected by 3′-O-5′-O phosphorothiodiestergroups; unless denoted by prefix “p-”, oligonucleotides are devoid of a5′-phosphate group on the 5′-most nucleotide; all oligonucleotides bear3′-OH on the 3′-most nucleotide.

TABLE 5b Silencing activity of modified dsRNA in monkey hepatocytesPrimary Position in IFN-α/ Cynomolgus Monkey human TNF-α HepatocytesName access.# Induction Sense Antisense ~IC50, nM AD-1a1 1091 Yes/Yesunmodified unmodified 0.07-0.2 AD-1a2 1091 No/No 2′OMe 2′OMe 0.07-0.2AD-1a3 1091 No/No Alt 2′F, Alt 2′F, 2′OMe 0.07-0.2 2′OMe AD-1a4 1091No/No 2′OMe 2′F all Py, 0.07-0.2 5′Phosphate AD-1a5 1091 No/No 2′F 2′Fall Py, 0.07-0.2 5′Phosphate AD-2a1 3530 No/No 2′OMe 2′OMe 0.07-0.2(3′UTR) AD-3a1  833 No/No 2′OMe 2′OMe  0.1-0.3 AD-ctrl N/A No/No 2′OMe2′OMe N/A (Luc.)

TABLE 6 dsRNA targeted to PCSK9: mismatches and modifications SEQ DuplexID # Strand NO: Sequence 5′ to 3′ AD-9680 S 1531uucuAGAccuGuuuuGcuudTsdT AS 1532 AAGcAAAAcAGGUCuAGAAdTsdT AD-3267 S 1535uucuAGAcCuGuuuuGcuuTsT AS 1536 AAGcAAAAcAGGUCuAGAATsT AD-3268 S 1537uucuAGAccUGuuuuGcuuTsT AS 1538 AAGcAAAAcAGGUCuAGAATsT AD-3269 S 1539uucuAGAcCUGuuuuGcuuTsT AS 1540 AAGcAAAAcAGGUCuAGAATsT AD-3270 S 1541uucuAGAcY1uGuuuuGcuuTsT AS 1542 AAGcAAAAcAGGUCuAGAATsT AD-3271 S 1543uucuAGAcY1UGuuuuGcuuTsT AS 1544 AAGcAAAAcAGGUCuAGAATsT AD-3272 S 1545uucuAGAccY1GuuuuGcuuTsT AS 1546 AAGcAAAAcAGGUCuAGAATsT AD-3273 S 1547uucuAGAcCY1GuuuuGcuuTsT AS 1548 AAGcAAAAcAGGUCuAGAATsT AD-3274 S 1549uucuAGAccuY1uuuuGcuuTsT AS 1550 AAGcAAAAcAGGUCuAGAATsT AD-3275 S 1551uucuAGAcCUY1uuuuGcuuTsT AS 1552 AAGcAAAAcAGGUCuAGAATsT AD-14676 S 1553UfuCfuAfgAfcCfuGfuUfuUfgCfuUfTsT AS 1554p-aAfgCfaAfaAfcAfgGfuCfuAfgAfaTsT AD-3276 S 1555UfuCfuAfgAfcCuGfuUfuUfgCfuUfTsT AS 1556p-aAfgCfaAfaAfcAfgGfuCfuAfgAfaTsT AD-3277 S 1557UfuCfuAfgAfcCfUGfuUfuUfgCfuUfTsT AS 1558p-aAfgCfaAfaAfcAfgGfuCfuAfgAfaTsT AD-3278 S 1559UfuCfuAfgAfcCUGfuUfuUfgCfuUfTsT AS 1560p-aAfgCfaAfaAfcAfgGfuCfuAfgAfaTsT AD-3279 S 1561UfuCfuAfgAfcY1uGfuUfuUfgCfuUfTsT AS 1562p-aAfgCfaAfaAfcAfgGfuCfuAfgAfaTsT AD-3280 S 1563UfuCfuAfgAfcY1UGfuUfuUfgCfuUfTsT AS 1564p-aAfgCfaAfaAfcAfgGfuCfuAfgAfaTsT AD-3281 S 1565UfuCfuAfgAfcCfY1GfuUfuUfgCfuUfTsT AS 1566p-aAfgCfaAfaAfcAfgGfuCfuAfgAfaTsT AD-3282 S 1567UfuCfuAfgAfcCY1GfuUfuUfgCfuUfTsT AS 1568p-aAfgCfaAfaAfcAfgGfuCfuAfgAfaTsT AD-3283 S 1569UfuCfuAfgAfcCfuY1uUfuUfgCfuUfTsT AS 1570p-aAfgCfaAfaAfcAfgGfuCfuAfgAfaTsT AD-3284 S 1571UfuCfuAfgAfcCUY1uUfuUfgCfuUfTsT AS 1572p-aAfgCfaAfaAfcAfgGfuCfuAfgAfaTsT AD-10792 S 459 GccuGGAGuuuAuucGGAATsTAS 460 UUCCGAAuAAACUCcAGGCTsT AD-3254 S 1573 GccuGGAGuY1uAuucGGAATsT AS1574 UUCCGAAuAAACUCcAGGCTsT AD-3255 S 1575 GccuGGAGUY1uAuucGGAATsT AS1576 UUCCGAAuAAACUCcAGGCTsT Strand: S/Sense; AS/Antisense U, C, A, G:corresponding ribonucleotide; T: deoxythymidine; u, c, a, g:corresponding 2′-O-methyl ribonucleotide; Uf, Cf, Af, Gf: corresponding2′-deoxy-2′-fluoro ribonucleotide; Y1 corresponds to DFT difluorotoluylribo(or deoxyribo)nucleotide; where nucleotides are written in sequence,they are connected by 3′-5′ phosphodiester groups; nucleotides withinterjected “s” are connected by 3′-O-5′-O phosphorothiodiester groups;unless denoted by prefix “p-”, oligonucleotides are devoid of a5′-phosphate group on the 5′-most nucleotide; all oligonucleotides bear3′-OH on the 3′-most nucleotide

TABLE 7 Sequences of unmodified siRNA flanking AD-9680 Target DuplexType Sequence (5′ to 3′) site SEQ ID NO: AD-22169-b1 senseCAGCCAACUUUUCUAGACCdTsdT 3520 1577 antis GGUCUAGAAAAGUUGGCUGdTsdT 35201578 AD-22170-b1 sense AGCCAACUUUUCUAGACCUdTsdT 3521 1579 antisAGGUCUAGAAAAGUUGGCUdTsdT 3521 1580 AD-22171-b1 senseGCCAACUUUUCUAGACCUGdTsdT 3522 1581 antis CAGGUCUAGAAAAGUUGGCdTsdT 35221582 AD-22172-b1 sense CCAACUUUUCUAGACCUGUdTsdT 3523 1583 antisACAGGUCUAGAAAAGUUGGdTsdT 3523 1584 AD-22173-b1 senseCAACUUUUCUAGACCUGUUdTsdT 3524 1585 antis AACAGGUCUAGAAAAGUUGdTsdT 35241586 AD-22174-b1 sense AACUUUUCUAGACCUGUUUdTsdT 3525 1587 antisAAACAGGUCUAGAAAAGUUdTsdT 3525 1588 AD-22175-b1 senseACUUUUCUAGACCUGUUUUdTsdT 3526 1589 antis AAAACAGGUCUAGAAAAGUdTsdT 35261590 AD-22176-b1 sense CUUUUCUAGACCUGUUUUGdTsdT 3527 1591 antisCAAAACAGGUCUAGAAAAGdTsdT 3527 1592 AD-22177-b1 senseUUUUCUAGACCUGUUUUGCdTsdT 3528 1593 antis GCAAAACAGGUCUAGAAAAdTsdT 35281594 AD-22178-b1 sense UUUCUAGACCUGUUUUGCUdTsdT 3529 1595 antisAGCAAAACAGGUCUAGAAAdTsdT 3529 1596 AD-22179-b1 senseUCUAGACCUGUUUUGCUUUdTsdT 3531 1597 antis AAAGCAAAACAGGUCUAGAdTsdT 35311598 AD-22180-b1 sense CUAGACCUGUUUUGCUUUUdTsdT 3532 1599 antisAAAAGCAAAACAGGUCUAGdTsdT 3532 1600 AD-22181-b1 senseUAGACCUGUUUUGCUUUUGdTsdT 3533 1601 antis CAAAAGCAAAACAGGUCUAdTsdT 35331602 AD-22182-b1 sense AGACCUGUUUUGCUUUUGUdTsdT 3534 1603 antisACAAAAGCAAAACAGGUCUdTsdT 3534 1604 AD-22183-b1 senseGACCUGUUUUGCUUUUGUAdTsdT 3535 1605 antis UACAAAAGCAAAACAGGUCdTsdT 35351606 AD-22184-b1 sense ACCUGUUUUGCUUUUGUAAdTsdT 3536 1607 antisUUACAAAAGCAAAACAGGUdTsdT 3536 1608 AD-22185-b1 senseCCUGUUUUGCUUUUGUAACdTsdT 3537 1609 antis GUUACAAAAGCAAAACAGGdTsdT 35371610 AD-22186-b1 sense CUGUUUUGCUUUUGUAACUdTsdT 3538 1611 antisAGUUACAAAAGCAAAACAGdTsdT 3538 1612 AD-22187-b1 senseUGUUUUGCUUUUGUAACUUdTsdT 3539 1613 antis AAGUUACAAAAGCAAAACAdTsdT 35391614 AD-22188-b1 sense GUUUUGCUUUUGUAACUUGdTsdT 3540 1615 antisCAAGUUACAAAAGCAAAACdTsdT 3540 1616 AD-22189-b1 senseUUUUGCUUUUGUAACUUGAdTsdT 3541 1617 antis UCAAGUUACAAAAGCAAAAdTsdT 35411618 AD-22190-b1 sense UUUGCUUUUGUAACUUGAAdTsdT 3542 1619 antisUUCAAGUUACAAAAGCAAAdTsdT 3542 1620 AD-22191-b1 senseUUGCUUUUGUAACUUGAAGdTsdT 3543 1621 antis CUUCAAGUUACAAAAGCAAdTsdT 35431622 AD-22192-b1 sense UGCUUUUGUAACUUGAAGAdTsdT 3544 1623 antisUCUUCAAGUUACAAAAGCAdTsdT 3544 1624 AD-22193-b1 senseGCUUUUGUAACUUGAAGAUdTsdT 3545 1625 antis AUCUUCAAGUUACAAAAGCdTsdT 35451626 AD-22194-b1 sense CUUUUGUAACUUGAAGAUAdTsdT 3546 1627 antisUAUCUUCAAGUUACAAAAGdTsdT 3546 1628 AD-22195-b1 senseUUUUGUAACUUGAAGAUAUdTsdT 3547 1629 antis AUAUCUUCAAGUUACAAAAdTsdT 35471630 AD-22196-b1 sense UUUGUAACUUGAAGAUAUUdTsdT 3548 1631 antisAAUAUCUUCAAGUUACAAAdTsdT 3548 1632 AD-22197-b1 senseUUGUAACUUGAAGAUAUUUdTsdT 3549 1633 antis AAAUAUCUUCAAGUUACAAdTsdT 35491634 AD-22198-b1 sense UGUAACUUGAAGAUAUUUAdTsdT 3550 1635 antisUAAAUAUCUUCAAGUUACAdTsdT 3550 1636 AD-22199-b1 senseGUAACUUGAAGAUAUUUAUdTsdT 3551 1637 antis AUAAAUAUCUUCAAGUUACdTsdT 35511638 AD-22200-b1 sense UAACUUGAAGAUAUUUAUUdTsdT 3552 1639 antisAAUAAAUAUCUUCAAGUUAdTsdT 3552 1640 AD-22201-b1 senseAACUUGAAGAUAUUUAUUCdTsdT 3553 1641 antis GAAUAAAUAUCUUCAAGUUdTsdT 35531642 AD-22202-b1 sense ACUUGAAGAUAUUUAUUCUdTsdT 3554 1643 antisAGAAUAAAUAUCUUCAAGUdTsdT 3554 1644 AD-22203-b1 senseCUUGAAGAUAUUUAUUCUGdTsdT 3555 1645 antis CAGAAUAAAUAUCUUCAAGdTsdT 35551646 AD-22204-b1 sense UUGAAGAUAUUUAUUCUGGdTsdT 3556 1647 antisCCAGAAUAAAUAUCUUCAAdTsdT 3556 1648 AD-22205-b1 senseUGAAGAUAUUUAUUCUGGGdTsdT 3557 1649 antis CCCAGAAUAAAUAUCUUCAdTsdT 35571650 AD-22206-b1 sense GAAGAUAUUUAUUCUGGGUdTsdT 3558 1651 antisACCCAGAAUAAAUAUCUUCdTsdT 3558 1652

TABLE 8 Sequences of modified siRNA flanking AD-9680 Duplex TypeSequence (5′ to 3′) Target SEQ ID NO: AD-22098-b1 sensecAGccAAcuuuucuAGAccdTsdT 3520 1653 antis GGUCuAGAAAAGUUGGCUGdTsdT 35201654 AD-22099-b1 sense AGccAAcuuuucuAGAccudTsdT 3521 1655 antisAGGUCuAGAAAAGUUGGCUdTsdT 3521 1656 AD-22100-b1 senseGccAAcuuuucuAGAccuGdTsdT 3522 1657 antis cAGGUCuAGAAAAGUUGGCdTsdT 35221658 AD-22101-b1 sense ccAAcuuuucuAGAccuGudTsdT 3523 1659 antisAcAGGUCuAGAAAAGUUGGdTsdT 3523 1660 AD-22102-b1 sensecAAcuuuucuAGAccuGuudTsdT 3524 1661 antis AAcAGGUCuAGAAAAGUUGdTsdT 35241662 AD-22103-b1 sense AAcuuuucuAGAccuGuuudTsdT 3525 1663 antisAAAcAGGUCuAGAAAAGUUdTsdT 3525 1664 AD-22104-b1 senseAcuuuucuAGAccuGuuuudTsdT 3526 1665 antis AAAAcAGGUCuAGAAAAGUdTsdT 35261666 AD-22105-b1 sense cuuuucuAGAccuGuuuuGdTsdT 3527 1667 antiscAAAAcAGGUCuAGAAAAGdTsdT 3527 1668 AD-22106-b1 senseuuuucuAGAccuGuuuuGcdTsdT 3528 1669 antis GcAAAAcAGGUCuAGAAAAdTsdT 35281670 AD-22107-b1 sense uuucuAGAccuGuuuuGcudTsdT 3529 1671 antisAGcAAAAcAGGUCuAGAAAdTsdT 3529 1672 AD-22108-b1 senseucuAGAccuGuuuuGcuuudTsdT 3531 1673 antis AAAGcAAAAcAGGUCuAGAdTsdT 35311674 AD-22109-b1 sense cuAGAccuGuuuuGcuuuudTsdT 3532 1675 antisAAAAGcAAAAcAGGUCuAGdTsdT 3532 1676 AD-22110-b1 senseuAGAccuGuuuuGcuuuuGdTsdT 3533 1677 antis cAAAAGcAAAAcAGGUCuAdTsdT 35331678 AD-22111-b1 sense AGAccuGuuuuGcuuuuGudTsdT 3534 1679 antisAcAAAAGcAAAAcAGGUCUdTsdT 3534 1680 AD-22112-b1 senseGAccuGuuuuGcuuuuGuAdTsdT 3535 1681 antis uAcAAAAGcAAAAcAGGUCdTsdT 35351682 AD-22113-b1 sense AccuGuuuuGcuuuuGuAAdTsdT 3536 1683 antisUuAcAAAAGcAAAAcAGGUdTsdT 3536 1684 AD-22114-b1 senseccuGuuuuGcuuuuGuAAcdTsdT 3537 1685 antis GUuAcAAAAGcAAAAcAGGdTsdT 35371686 AD-22115-b1 sense cuGuuuuGcuuuuGuAAcudTsdT 3538 1687 antisAGUuAcAAAAGcAAAAcAGdTsdT 3538 1688 sense uGuuuuGcuuuuGuAAcuudTsdT 35391689 antis AAGUuAcAAAAGcAAAAcAdTsdT 3539 1690 AD-22116-b1 senseGuuuuGcuuuuGuAAcuuGdTsdT 3540 1691 antis cAAGUuAcAAAAGcAAAACdTsdT 35401692 AD-22117-b1 sense uuuuGcuuuuGuAAcuuGAdTsdT 3541 1693 antisUcAAGUuAcAAAAGcAAAAdTsdT 3541 1694 AD-22118-b1 senseuuuGcuuuuGuAAcuuGAAdTsdT 3542 1695 antis UUcAAGUuAcAAAAGcAAAdTsdT 35421696 AD-22119-b1 sense uuGcuuuuGuAAcuuGAAGdTsdT 3543 1697 antisCUUcAAGUuAcAAAAGcAAdTsdT 3543 1698 AD-22120-b1 senseuGcuuuuGuAAcuuGAAGAdTsdT 3544 1699 antis UCUUcAAGUuAcAAAAGcAdTsdT 35441700 AD-22121-b1 sense GcuuuuGuAAcuuGAAGAudTsdT 3545 1701 antisAUCUUcAAGUuAcAAAAGCdTsdT 3545 1702 AD-22122-b1 sensecuuuuGuAAcuuGAAGAuAdTsdT 3546 1703 antis uAUCUUcAAGUuAcAAAAGdTsdT 35461704 AD-22123-b1 sense uuuuGuAAcuuGAAGAuAudTsdT 3547 1705 antisAuAUCUUcAAGUuAcAAAAdTsdT 3547 1706 AD-22124-b1 senseuuuGuAAcuuGAAGAuAuudTsdT 3548 1707 antis AAuAUCUUcAAGUuAcAAAdTsdT 35481708 AD-22125-b1 sense uuGuAAcuuGAAGAuAuuudTsdT 3549 1709 antisAAAuAUCUUcAAGUuAcAAdTsdT 3549 1710 AD-22126-b1 senseuGuAAcuuGAAGAuAuuuAdTsdT 3550 1711 antis uAAAuAUCUUcAAGUuAcAdTsdT 35501712 AD-22127-b1 sense GuAAcuuGAAGAuAuuuAudTsdT 3551 1713 antisAuAAAuAUCUUcAAGUuACdTsdT 3551 1714 AD-22128-b1 senseuAAcuuGAAGAuAuuuAuudTsdT 3552 1715 antis AAuAAAuAUCUUcAAGUuAdTsdT 35521716 AD-22129-b1 sense AAcuuGAAGAuAuuuAuucdTsdT 3553 1717 antisGAAuAAAuAUCUUcAAGUUdTsdT 3553 1718 AD-22130-b1 senseAcuuGAAGAuAuuuAuucudTsdT 3554 1719 antis AGAAuAAAuAUCUUcAAGUdTsdT 35541720 AD-22131-b1 sense cuuGAAGAuAuuuAuucuGdTsdT 3555 1721 antiscAGAAuAAAuAUCUUcAAGdTsdT 3555 1722 AD-22132-b1 senseuuGAAGAuAuuuAuucuGGdTsdT 3556 1723 antis CcAGAAuAAAuAUCUUcAAdTsdT 35561724 AD-22133-b1 sense uGAAGAuAuuuAuucuGGGdTsdT 3557 1725 antisCCcAGAAuAAAuAUCUUcAdTsdT 3557 1726 AD-22134-b1 senseGAAGAuAuuuAuucuGGGudTsdT 3558 1727 antis ACCcAGAAuAAAuAUCUUCdTsdT 35581728

TABLE 9 Single dose treatment of HeLa cells with siRNA flanking AD-9680% message % message remaining remaining Duplex ID 0.1 nM SD 0.1 nM 10 nMSD 10 nM AD-22098-b1 10.6 1.9 9.2 3.7 AD-22098-b1 7.7 1.7 7.9 0.7AD-22099-b1 21.3 4.5 27.4 7.2 AD-22099-b1 25.9 2.4 29.6 9.1 AD-22100-b158.6 9.6 35.8 11.1 AD-22100-b1 62.5 0.3 27.4 3.5 AD-22101-b1 21.9 3.812.9 1.4 AD-22101-b1 19.3 0.3 9.7 1.3 AD-22102-b1 6.6 0.1 7.7 3.3AD-22103-b1 8.7 0.0 8.2 1.3 AD-22104-b1 7.6 0.2 8.5 2.8 AD-22105-b1 13.41.0 8.1 2.3 AD-22106-b1 59.1 0.4 35.4 4.6 AD-22107-b1 9.1 0.8 8.4 3.7AD-22108-b1 8.8 0.9 6.2 1.7 AD-22109-b1 9.8 0.9 8.2 1.7 AD-22110-b1 24.81.7 15.3 5.9 AD-22111-b1 8.3 0.7 6.2 1.7 AD-22112-b1 15.1 0.0 10.3 2.9AD-22113-b1 10.9 0.6 10.0 2.0 AD-22114-b1 8.9 1.1 7.3 1.3 AD-22115-b15.3 0.8 3.7 0.7 AD-22116-b1 58.1 0.4 34.5 7.3 AD-22117-b1 19.9 0.9 12.22.9 AD-22118-b1 5.3 0.0 4.4 1.0 AD-22119-b1 8.6 1.9 5.8 2.3 AD-22120-b17.2 0.8 5.8 2.4 AD-22121-b1 7.3 0.9 6.4 2.1 AD-22122-b1 32.5 2.5 18.16.3 AD-22123-b1 14.7 0.8 16.7 7.0 AD-22124-b1 12.8 1.9 10.5 5.3AD-22125-b1 7.4 0.6 9.0 4.6 AD-22126-b1 12.8 0.4 16.4 7.3 AD-22127-b18.8 0.5 9.6 5.0 AD-22128-b1 9.9 0.2 12.4 5.9 AD-22129-b1 85.9 10.3 94.949.8 AD-22130-b1 5.6 1.0 6.2 4.1 AD-22131-b1 26.9 8.4 12.9 7.3AD-22132-b1 78.5 18.5 67.5 34.1 AD-22133-b1 26.4 7.1 15.0 6.7AD-22134-b1 26.9 0.1 22.4 6.5 AD-22169-b1 7.3 0.6 6.0 1.5 AD-22169-b17.0 1.1 6.1 1.3 AD-22170-b1 9.3 1.6 7.2 1.8 AD-22170-b1 9.7 1.1 11.2 1.0AD-22171-b1 7.1 2.3 4.5 0.2 AD-22171-b1 6.5 1.9 4.4 2.8 AD-22172-b1 7.21.1 7.6 3.7 AD-22172-b1 7.0 0.4 7.0 2.4 AD-22173-b1 15.7 12.5 5.9 0.1AD-22174-b1 8.9 2.7 6.4 0.9 AD-22175-b1 10.7 4.3 7.9 2.4 AD-22176-b1 9.60.8 8.4 3.1 AD-22177-b1 38.9 5.9 21.4 1.2 AD-22178-b1 6.5 0.5 5.6 0.9AD-22179-b1 7.0 0.8 5.9 0.1 AD-22180-b1 7.3 3.7 7.2 1.6 AD-22181-b1 11.10.9 10.0 1.0 AD-22182-b1 5.4 1.4 4.0 1.5 AD-22183-b1 3.8 0.4 2.9 0.4AD-22184-b1 5.1 0.2 3.7 0.7 AD-22185-b1 5.7 0.6 5.0 1.5 AD-22186-b1 5.30.3 5.7 1.0 AD-22187-b1 5.3 1.2 5.3 1.4 AD-22188-b1 12.6 2.6 11.6 0.2AD-22189-b1 5.2 0.5 4.5 1.8 AD-22190-b1 4.7 1.3 3.4 1.1 AD-22191-b1 10.50.6 7.9 0.9 AD-22192-b1 6.9 2.2 5.8 3.5 AD-22193-b1 7.5 1.5 5.2 0.6AD-22194-b1 8.0 1.4 6.5 1.9 AD-22195-b1 7.0 1.9 4.9 2.3 AD-22196-b1 5.40.0 3.8 0.9 AD-22197-b1 6.6 0.4 5.2 1.2 AD-22198-b1 7.3 0.8 8.5 2.4AD-22199-b1 5.5 0.7 4.2 1.2 AD-22200-b1 11.0 0.5 12.5 3.1 AD-22201-b144.0 3.1 47.3 8.3 AD-22202-b1 9.0 1.2 7.2 0.9 AD-22203-b1 12.5 0.0 12.72.2 AD-22204-b1 57.1 5.2 50.2 10.2 AD-22205-b1 27.0 0.4 24.5 0.0AD-22206-b1 13.9 1.1 11.4 1.3 AD-9680 7.1 ND 9.3 ND

TABLE 10 IC50 in HeLa cells using siRNA flanking AD-9680 Rep1 Rep2 IC50IC50 Average IC50 Duplex Name (pM) (pM) (pM) AD-22098 6.0 6.7 6.4AD-22099 25.0 37.8 31.4 AD-22101 66.5 81.9 74.2 AD-22102 2.3 1.5 1.9AD-22103 6.3 1.2 3.8 AD-22104 2.2 1.4 1.8 AD-22105 13.3 0.1 6.7 AD-221072.2 0.9 1.6 AD-22108 2.3 2.0 2.1 AD-22109 5.5 6.3 5.9 AD-22110 59.1 42.250.7 AD-22111 9.1 8.2 8.7 AD-22112 25.8 31.0 28.4 AD-22113 4.2 4.4 4.3AD-22114 6.9 4.0 5.5 AD-22115 3.0 2.2 2.6 AD-22117 56.0 37.6 46.8AD-22118 2.9 1.7 2.3 AD-22119 6.7 0.0 3.4 AD-22120 2.0 1.2 1.6 AD-221212.1 4.1 3.1 AD-22122 203.3 156.3 179.8 AD-22123 33.1 50.7 41.9 AD-2212418.8 13.1 15.9 AD-22125 3.3 2.6 3.0 AD-22126 17.9 18.5 18.2 AD-2212711.1 4.3 7.7 AD-22128 14.6 3.3 8.9 AD-22130 1.7 0.3 1.0 AD-22131 172.559.6 116.0 AD-22133 94.6 57.2 75.9 AD-22134 113.0 81.3 97.2 AD-9680 3.82.4 3.1

1. A composition comprising a nucleic acid lipid particle comprising adouble-stranded ribonucleic acid (dsRNA) for inhibiting the expressionof a human PCSK9 gene in a cell, wherein: the nucleic acid lipidparticle comprises a lipid formulation comprising 45-65 mol % of acationic lipid, 5 mol % to about 10 mol %, of a non-cationic lipid,25-40 mol % of a sterol, and 0.5-5 mol % of a PEG or PEG-modified lipid,the dsRNA consists of a sense strand and an antisense strand, and thesense strand comprises a first sequence and the antisense strandcomprises a second sequence complementary to at least 15 contiguousnucleotides of a nucleotide sequence of a target sequence of a dsRNAfound in Table 1a, Table 2a, Table 5a, Table 6, Table 7, Table 8,wherein the first sequence is complementary to the second sequence andwherein the dsRNA is between 15 and 30 base pairs in length.
 2. Thecomposition of claim 1, wherein the cationic lipid comprises MC3(((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate).3. The composition of claim 2, wherein the cationic lipid comprises MC3and the lipid formulation is selected from the group consisting of:LNP11 MC3/DSPC/Cholesterol/PEG-DMG 50/10/38.5/1.5 LNP14MC3/DSPC/Cholesterol/PEG-DMG 40/15/40/5 LNP15MC3/DSPC/Cholesterol/PEG-DSG/GalNAc-PEG-DSG 50/10/35/4.5/0.5 LNP16MC3/DSPC/Cholesterol/PEG-DMG 50/10/38.5/1.5 LNP17MC3/DSPC/Cholesterol/PEG-DSG 50/10/38.5/1.5 LNP18MC3/DSPC/Cholesterol/PEG-DMG 50/10/38.5/1.5 LNP19MC3/DSPC/Cholesterol/PEG-DMG 50/10/35/5 LNP20MC3/DSPC/Cholesterol/PEG-DPG 50/10/38.5/1.5

4.-9. (canceled)
 10. The composition of claim 1, wherein the sensestrand comprises SEQ ID NO:1227 and the antisense strand comprises SEQID NO:1228.
 11. (canceled)
 12. (canceled)
 13. The composition of claim1, wherein the dsRNA comprises at least one modified nucleotide.
 14. Thecomposition of claim 13, wherein the modified nucleotide is chosen fromthe group of: a 2′-O-methyl modified nucleotide, a nucleotide comprisinga 5′-phosphorothioate group, and a terminal nucleotide linked to acholesteryl derivative or dodecanoic acid bisdecylamide group.
 15. Thecomposition of claim 13, wherein the modified nucleotide is chosen fromthe group of: a 2′-deoxy-2′-fluoro modified nucleotide, a2′-deoxy-modified nucleotide, a locked nucleotide, an abasic nucleotide,2′-amino-modified nucleotide, 2′-alkyl-modified nucleotide, morpholinonucleotide, a phosphoramidate, and a non-natural base comprisingnucleotide.
 16. The composition of claim 1, wherein the dsRNA comprisesat least one 2′-O-methyl modified ribonucleotide and at least onenucleotide comprising a 5′-phosphorothioate group.
 17. The compositionof claim 1, wherein each strand of the dsRNA is 19-23 bases in length.18. The composition of claim 1, wherein each strand of the dsRNA is21-23 bases in length.
 19. The composition of claim 1, wherein eachstrand of the dsRNA is 21 bases in length.
 20. The composition of claim1, further comprising a lipoprotein.
 21. The composition of claim 1,further comprising apolipoprotein E (ApoE).
 22. The composition of claim21, wherein the dsRNA is conjugated to a lipophile.
 23. The compositionof claim 22, wherein the lipophile is cholesterol.
 24. The compositionof claim 21, wherein the ApoE is reconstituted with at least oneamphiphilic agent.
 25. The composition of claim 24, wherein theamphiphilic agent is a phospholipid.
 26. The composition of claim 24,wherein the amphilic agent is a phospholipid selected from the groupconsisting of dimyristoyl phosphatidyl choline (DMPC),dioleoylphosphatidylethanolamine (DOPE),palmitoyloleoylphosphatidylcholine (POPC), egg phosphatidylcholine(EPC), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine(DOPC), dipalmitoylphosphatidylcholine (DPPC),dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol(DPPG), -phosphatidylethanolamine (POPE),dioleoyl-phosphatidylethanolamine4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), andcombinations thereof.
 27. The composition of claim 25, wherein the ApoEis reconstituted high density lipoprotein (rHDL). 28.-33. (canceled) 34.A method for inhibiting the expression of PCSK9 in a cell comprisingadministering the composition of claim 1 to the cell. 35.-43. (canceled)